Quantitative High-performance Liquid Chromatography Analysis Research Articles

Forsythia suspensa fruit extracts and the constituent matairesinol confer anti-allergic effects in an allergic dermatitis mouse model

Ethnopharmacological relevanceForsythia suspensa is used in traditional medicine to treat inflammation. To clarify the anti-inflammatory and anti-allergic effects of F. suspensa fruits, we determined the therapeutic effects of crude extract, fractions, and a constituent from F. suspensa fruits on a murine atopic dermatitis (AD) model. Materials and methodsWe investigated the inhibitory effects of F. suspensa extract (FSE), extract fractions, and the constituent matairesinol on histamine release from MC/9 mast cells activated by compound 48/80 and the development of AD-like skin lesions and symptoms in NC/Nga mice exposed to Dermatophagoides farinae (mite) extract. High performance liquid chromatography (HPLC) analysis of FSE and its fractions were evaluated using matairesinol standard. ResultsFSE, FSE methylene chloride fraction (FSE-MC), and FSE water fraction (FSE-water) inhibited compound 48/80-induced histamine release from MC/9 mast cells. Topical application of FSE or FSE-MC to NC/Nga mice exposed to Dermatophagoides farinae suppressed the development of AD-like skin lesions. Quantitative HPLC analysis of FSE and FSE-MC identified the presence of matairesinol. Topical application of matairesinol to NC/Nga mice effectively reduced AD symptoms, inhibited inflammatory cell infiltration, and lowered immunoglobulin E levels in serum. Further study demonstrated that DfE-induced changes in IL-4 and IFN-γ mRNA expression in the ears of NC/Nga mice were reversed by matairesinol application. ConclusionsThese results indicate that the F. suspensa and its constituent matairesinol might be a therapeutic candidate for treating allergic inflammatory disorders such as AD.

Quick and selective extraction of Z-ligustilide from Angelica sinensis using magnetic multiwalled carbon nanotubes.

A facile and highly efficient magnetic solid-phase extraction method has been developed for Z-ligustilide, the major therapeutic agent in Angelica sinensis. The solid-phase adsorbent material used was prepared by conjugating carbon nanotubes with magnetic Fe3 O4 nanoparticles via a hydrothermal reaction. The magnetic material showed a high affinity toward Z-ligustilide due to the π-π stacking interaction between the carbon nanotubes and Z-ligustilide, allowing a quick and selective exaction of Z-ligustilide from complex sample matrices. Factors influencing the magnetic solid-phase extraction such as the amount of the added adsorbent, adsorption and desorption time, and desorption solvent, were investigated. Due to its high extraction efficiency, this method was proved highly useful for sample cleanup/enrichment in quantitative high-performance liquid chromatography analysis. The proposed method had a linear calibration curve (R(2) = 0.9983) over the concentration between 4 ng/mL and 200 μg/mL Z-ligustilide. The accuracy of the method was determined by the recovery, which was from 92.07 to 104.02%, with the relative standard deviations >4.51%.

Monthly metabolic changes and PLS prediction of carotenoid content of citrus fruit by combined Fourier transform infrared spectroscopy and quantitative HPLC analysis

This work examined the potential of Fourier transform infrared spectroscopy (FT-IR) spectroscopy and high-performance liquid chromatography (HPLC) analysis in evaluating metabolic changes during ripening of citrus fruit. Further, the feasibility of prediction modeling of carotenoid content by multivariate statistical analysis combined with FT-IR spectral and HPLC data was examined without additional HPLC analysis. FT-IR spectra of citrus (Citrus unshiu Marc. cv. Miyagawa) fruit peels and flesh were measured at monthly intervals of fruit development. Quantification of carotenoids from the fruit was confirmed by quantitative HPLC analysis. The most remarkable evolution of FT-IR spectral decrease during ripening of fruit was found in the amide region (1500–1700 cm−1), whereas there was an increase in the carbohydrate region (1000–1200 cm−1). The evolution of different FT-IR spectral bands was related to fruit constituents, including organic acids, carbohydrates, carotenoids, and phenolic compounds. Significant qualitative changes in the carotenoid pattern included an increase in β-cryptoxanthin and decrease in lutein content during citrus fruit development. The content of antheraxanthin (R 2 = 0.9117), β-carotene (R 2 = 0.8816), β-cryptoxanthin (R 2 = 0.8856), and violaxanthin (R 2 = 0.7305) from peels of citrus fruit could be predicted from FT-IR spectral data using partial least square (PLS) regression modeling. Considering the results of PLS-discriminant analysis (PLS-DA) of FT-IR spectral data and PLS regression modeling of carotenoid content, FT-IR in combination with multivariate analysis enables not only discrimination of metabolic variation during fruit development, but also prediction of carotenoid content from citrus fruit.

Effect of Ganoderma applanatum mycelium extract on the inhibition of adipogenesis in 3T3-L1 adipocytes.

Ganoderma applanatum (GA) and related fungal species have been used for over 2000 years in China to prevent and treat various human diseases. However, there is no critical research evaluating the functionality of GA grown using submerged culture technology. This study aimed to evaluate the effects of submerged culture GA mycelium (GAM) and its active components (protocatechualdehyde [PCA]) on preadipocyte differentiation of 3T3-L1 cells. Mouse-derived preadipocyte 3T3-L1 cells were treated with differentiation inducers in the presence or absence of GAM extracts. We determined triglyceride accumulations, glycerol-3-phosphate dehydrogenase (GPDH) activities, and differentiation makers. PCA, the active component of GAM extract, was also used to treat 3T3-L1 cells. The MTT assay showed that the GAM extract (0.01-1 mg/mL) was not toxic to 3T3-L1 preadipocyte. Treatment of cells with GAM extracts and its active components significantly decreased the GPDH activity and lipid accumulation, a marker of adipogenesis, in a dose-dependent manner. Western blot analysis results showed that the protein expression levels of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), and sterol regulatory element-binding protein 1 (SREBP1) were inhibited by the GAM extract. In addition, adipogenic-specific genes such as perilipin, fatty acid synthase (FAS), fatty acid transport protein 1 (FATP1), and fatty acid-binding protein 4 (FABP4) decreased in a dose-dependent manner. Quantitative high-performance liquid chromatography analysis showed that the GAM extract contained 1.14 mg/g PCA. GAM extracts suppressed differentiation of 3T3-L1 preadipocytes, in part, through altered regulation of PPARγ, C/EBPα, and SREBP1. These results suggest that GAM extracts and PCA may suppress adipogenesis by inhibiting differentiation of preadipocytes.

Identification of Phenolic Compounds in Processed Cranberries by HPLC Method

Cranberries contain significant amounts of flavonoids and polyphenolic compounds that provide health benefits including antioxidants functions, protection against cardiovascular diseases and anti-adhesive properties that help to prevent urinary tract infections and stomach ulcers. In this study, phenolic compounds in different cranberry products were extracted using methanol and acetone. The phenolic extracts were then analysed using high performance liquid chromatography (HPLC). The results showed that the different cranberry products had different phenolic types (including tannic acid, (+) catechin, (-) epicatechin, procyanidin A2 and p-coumaric acid) in different amounts. Moreover, the results varied depending on the extraction methods. Generally, the methanol extracts had higher total phenolic in comparison to acetone extracts of all the different cranberry products studied. For methanol extraction, frozen cranberry had the highest total phenolic content (616.65 ± 0.14 mg/g), followed by sauces (360.80 ± 0.09 mg/g), then 17% juices (237.84 ± 0.03 mg/ml) and lastly, dry cranberry (142.13 ± 0.05 mg/g). However, for acetone extraction, the total phenolic content were in the order: frozen (609.92 ± 0.12 mg/g) > 17% juices (269.27 ± 0.06 mg/ml) > sauce (94.03 ± 0.06 mg/g) > dry cranberry (0.57 ± 0.01 mg/g). The results of the present study showed that methanol serves as a better solvent than acetone for extracting phenolic compounds for both qualitative and quantitative HPLC analysis of phenolic compounds in cranberry products.

Bioassay-Guided Isolation and HPLC Determination of Bioactive Compound That Relate to the Antiplatelet Activity (Adhesion, Secretion, and Aggregation) fromSolanum lycopersicum

In seeking the functionality of foodstuff applicable to medicine, ripe tomato fruits were found to show an antiplatelet activity. Therefore, the bioactive compound was isolated, structurally identified, and studied for an inhibitory effects on platelet adhesion, secretion, and aggregation. The concentration of adenosine in ripe tomato fruits (pulp and skin extracts) and its processing by-products (paste and pomace) was determined by reversed-phase high-performance liquid chromatography (HPLC). According to platelet aggregation inhibition induced by ADP, the total extract residual was fractionated by liquid-liquid separation, obtaining aqueous, ethyl acetate and petroleum ether extracts. The aqueous extract was subjected to repeated permeation over sephadex LH-20 and semipreparative TLC. The isolate finally obtained was identified as adenosine on the basis of ESI-MS, 1H NMR, HPLC, and UV spectra. Adenosine concentration dependently (2.3–457 μM) platelet aggregation inhibited induced by ADP. Also, adenosine present inhibition of platelet secretion and thrombus formation under flow conditions. The quantitative HPLC analysis revealed significant amounts of adenosine in ripe tomato fruits and its processing by-products. From these results, extracts/fractions of ripe tomato fruits and their processing by-products may be referred to as functional food and functional ingredients containing a compound that inhibits platelet function with a potent preventive effect on thrombus formation, as those that occur in stroke.

Simple Chromatographic Method for Simultaneous Analyses of Phosphatidylcholine, Lysophosphatidylcholine, and Free Fatty Acids

This study describes a simple chromatographic method for the simultaneous analyses of phosphatidylcholine (PC) and its hydrolytic degradation products: lysophosphatidylcholine (LPC) and free fatty acids (FFA). Quantitative determination of PC, LPC, and FFA is essential in order to assure safety and to accurately assess the shelf life of phospholipid-containing products. A single-run normal-phase high-performance liquid chromatography (HPLC) with evaporative light scattering detector has been developed. The method utilizes an Allsphere silica analytical column and a gradient elution with mobile phases consisting of chloroform: chloroform-methanol (70:30%, v/v) and chloroform-methanol-water-ammonia (45:45:9.5:0.5%, v/v/v/v). The method adequately resolves PC, LPC, and FFA within a run time of 25 min. The quantitative analysis of PC and LPC has been achieved with external standard method. The free fatty acids were analyzed as a group using linoleic acid as representative standard. Linear calibration curves were obtained for PC (1.64-16.3 μg, r(2) = 0.9991) and LPC (0.6-5.0 μg, r(2) = 0.9966), while a logarithmic calibration curve was obtained for linoleic acid (1.1-5.8 μg, r(2) = 0.9967). The detection and quantification limits of LPC and FFA were 0.04 and 0.1 μg, respectively. As a means of validating the applicability of the assay to pharmaceutical products, PC liposome was subjected to alkaline hydrolytic degradation. Quantitative HPLC analysis showed that 97% of the total mass balance for PC could be accounted for in liposome formulation. The overall results show that the HPLC method could be a useful tool for chromatographic analysis, stability studies, and formulation characterization of phospholipid-based pharmaceuticals.

Quantitative HPLC Analysis of Riboflavin and Aromatic Amino Acids in Three Forms of Shrimp Hydrolysates

This paper presents a high performance liquid chromatography (HPLC) method for the determination of riboflavin and free aromatic amino acids. To determine riboflavin, the method includes an acid hydrolysis (HCl 0.1 N) followed by an enzymatic digestion and a protein precipitation with trichloroacetic acid. An analytical column, Chrom SEP SS C18 5 µm (4.6 mm × 150 mm) was used with an isocratic flow using a mobile phase consisting of 5 mM ammonium acetate-methanol (72:28, vv−1), at a flow rate of 1.0 mL min−1 to 28°C. The method showed adequate linearity, repeatability, reproducibility, accuracy, and limits of detection. The method was used to quantify the cited analytes in three different forms of hydrolysate obtained from fermented shrimp remnants.

Quantitative high-performance liquid chromatography analysis of the pool levels of undecaprenyl phosphate and its derivatives in bacterial membranes

Undecaprenyl phosphate is the essential lipid involved in the transport of hydrophilic motifs across the bacterial membranes during the synthesis of cell wall polymers such as peptidoglycan. A HPLC procedure was developed for the quantification of undecaprenyl phosphate and its two derivatives, undecaprenyl pyrophosphate and undecaprenol. During the exponential growth phase, the pools of undecaprenyl phosphate and undecaprenyl pyrophosphate were ca. 75 and 270 nmol/g of cell dry weight, respectively, in Escherichia coli, and ca. 50 and 150 nmol/g, respectively, in Staphylococcus aureus. Undecaprenol was detected in S. aureus (70 nmol/g), but not in E. coli (<1 nmol/g).

Simple and rapid quantitative high-performance liquid chromatographic analysis of plasma amino acids

A simple and rapid high performance liquid chromatographic method for the determination of plasma amino acids was developed. The method uses minimal sample volume and automated online precolumn derivitization of amino acids with o-phthalaldehyde and fluorescent detection. Amino acids are separated by a simplified gradient without column heating. The assay is linear from 5 to 1000 μmol/L for all amino acids. Recovery of amino acids was between 91 and 108%, intra-assay coefficient of variation (CV) was 1–7%, and inter-assay CV was 2–12%. The simple sample preparation and minimal sample volume make the method useful for the quantitation of amino acids in both patient and experimental animal samples.

Detection of a large genomic deletion in the pancreatic secretory trypsin inhibitor (SPINK1) gene

Mutations and polymorphisms in the SPINK1 gene, which encodes trypsin's physiological inhibitor, pancreatic secretory trypsin inhibitor, have been found to be associated with chronic pancreatitis. However, to date, all currently reported SPINK1 variants are either single-nucleotide substitutions or microinsertions/deletions. It is possible that large genomic rearrangements at this locus may underlie certain cases of chronic pancreatitis. However, such events, if indeed they exist, may have been overlooked by conventional PCR-based techniques. Here we attempted to screen all four exons as well as the promoter region of the SPINK1 gene for large genomic deletions by means of quantitative high-performance liquid chromatography analysis. Of the 47 pancreatitis families (not carrying any known PRSS1, SPINK1 and CFTR variants/mutations after screening the coding regions by our previously established denaturing high-performance liquid chromatography methods), one family was suggested to carry a large genomic deletion in the SPINK1 gene. The aberrant chromosomal junction was encapsulated by long-range PCR and the breakpoints were determined by direct sequencing of the rearranged fragment. A 2-bp short direct repeat was present at the deletion breakpoints; this simple deletion (c.1-320_c.55+961del1336 bp) can thus in principle be explained by replication slippage. Identification of this lesion has not only expanded the SPINK1 mutational spectrum but also served to identify a novel mutational mechanism causing chronic pancreatitis.

Two‐Stage Fractionation of a Mixture of 10 Pesticides by TLC and HPLC

Relationships between R F values and mobile‐phase composition have been determined for 10 pesticides in normal‐phase (NP) systems, which have enabled choosing optimum systems for preliminary fractionation of the multicomponent mixture of pesticides by zonal micropreparative thin‐layer chromatography (TLC). The 10‐component mixture was applied from the edge of the silica layer and developed with ethyl acetate–diisopropyl ether (10∶90, v/v). The zones were detected in UV light at λ = 254 nm. The separated eight simpler fractions were applied to an octadecyl silica layer wettable with water (TLC, RP‐18W) and rechromatographed. The separated eight simpler fractions were also applied to a cyanopropyl‐bonded silica layer and developed with (NP) and reverse‐phase (RP) systems. The plates were scanned and videoscanned, furnishing real pictures of the plates showing complete separation of the fractions on the RP‐18W plate with the RP and NP systems on cyanopropyl silica layer. The simpler fractions were also separated on a cyanopropyl and octadecyl silica high performance liquid chromatography (HPLC) columns. Preparative separation of the complex mixture by TLC on silica (non‐aqueous eluent, NP system), combined with TLC or/and HPLC (aqueous eluent, RP system) or HPLC (NP system), gives a good perspective of full separation of the simpler fractions in the second stage, by using the two methods with the possibility of full quantitative TLC and HPLC analysis.

Simultaneous identification/determination system for phentolamine and sildenafil as adulterants in soft drinks advertising roborant nutrition

An easily available, simultaneous identification/determination procedure for phentolamine (PHE) and sildenafil (SIL) in adulterated dietary supplements was established by using a combination of three different analytical methods; thin-layer chromatography (TLC), liquid chromatography–mass spectrometry (LC/MS) and a high-performance liquid chromatography (HPLC)/photo-diode-array. The sample solution for TLC was applied to silica gel 60 F 254 plates with chloroform/ammonia solution (28)/methanol (70:5:3, lower layer) and chloroform/diethylamine/methanol (15:3:2) as the developing solvent. Spots were located under UV radiation at 254 nm. Mass spectra of PHE and SIL by LC/MS were investigated with electrospray ionization (ESI) interface, under both positive and negative ion mode. The HPLC analysis was performed on a column of Wakosil 5C18 ( 4.6 mm×150 mm, 5 μm) with water/methanol/acetonitrile/triethylamine (580:250:170:1) adjusted with phosphoric acid to pH 3.0 as the mobile phase, and the effluent was monitored with a photo-diode-array detector. Quantitative HPLC analysis of PHE and SIL were detected at 280 nm. When this procedure was applied to commercial soft drinks, PHE and SIL were identified and determined at a concentration of 17 mg PHE and 44 mg SIL per bottle, respectively. The procedure described here is available for the screening of PHE and SIL in adulterated supplements.

Extraction and Quantitative HPLC Analysis of Coumarin in Hydroalcoholic Extracts of Mikania glomerata Spreng: ("guaco") Leaves

Methods for preparation of hydroalcoholic extracts of "guaco" (Mikania glomerata Spreng.) leaves were compared: maceration, maceration under sonication, infusion and supercritical fluid extraction. Evaluation of these methods showed that maceration under sonication had the best results, when considering the ratio extraction yield/extraction time. A high performance liquid chromatography (HPLC) procedure for the determination of coumarin in these hydroalcoholic extracts of "guaco" leaves is described. The HPLC method is shown to be sensitive and reproducible.

On-line dialysis and quantitative high-performance liquid chromatography analysis of iodixanol in human, rat and monkey plasma

A fully automated HPLC method for analysing the non-ionic X-ray contrast agent iodixanol in plasma samples, using on-line dialysis for sample preparation, was developed. Optimal conditions were obtained with a static dialysis donor solution of 110 μl and 4 ml of recipient solution (dialysate) pulsed onto a trace enrichment column, giving maximum 55% dialysis efficiency in less than the chromatographic run time of 20 min. Hence, one sample could be dialysed during the analysis of the previous. The maximum load of iodixanol on the trace enrichment column was 3.75 mg. Validation showed that the method was selective for iodixanol, sensitive down to 84 pmol/ml and had a high precision over a linear range up to 320 nmol/ml. Although developed for iodixanol, the method can easily be modified and applied to other substances with similar properties, i.e., substances having low protein binding and high water solubility, but strong enough stationary phase affinity to be retained by an appropriate trace enrichment column.

Structural Determination of Subsidiary Colors in Food Red No. 104 and Their Contents in Commercial Products

Subsidiary colors (P1 and P2) in commercial food red No. 104 (R104) were isolated as acidic forms by preparative high performance liquid chromatography (HPLC). Their structures were characterized on the basis of physicochemical evidence. The structures of P1 and P2 are 4′, 5′-dibromo-4, 5, 6, 7-tetrachloro-3′, 6′-dioxidospiro[isobenzofuran-1(3H), 9′-[9H]xanthen]-3-one and 2′, 4′, 5′-tribromo-4, 5, 6, 7-tetrachloro-3′, 6′-dioxidospiro[isobenzofuran-1(3H), 9′-[9H]xanthen]-3-one, respectively. Quantitative HPLC analyses of P1 and P2 were performed on commercial R 104 (9 samples from 4 makers). P2 was contained in all the samples in the range from 0.08% to 5.21%, while P1 was not detected in 5 samples and the highest content was 0.06%.

Quantitative HPLC Analysis of a Psychotherapeutic Medication: Simultaneous Determination of Amitriptyline Hydrochloride and Perphenazine

A quantitative high-performance liquid chromatography (HPLC) laboratory experiment which entails the isocratic separation and simultaneous determination of the two active components of a commercial antipsychotic tablet has been developed. The prescription formulation used in this experiment contains amitriptyline hydrochloride (a tricyclic antidepressant) and perphenazine (a tranquilizer). Our experiment makes use of a straightforward HPLC separation on a cyanopropyl-packed column with an acetonitrile:methanol:aqueous monopotassium phosphate mobile phase pumped at a flow rate of 2.0 mL/min. Analytes are detected by UV absorbance at 215 nm. These conditions yield highly symmetrical and well-resolved peaks in less than 5 min after the injection of a mixture.In the experiment, students are given amitriptyline hydrochloride-perphenazine tablets without the manufacturer's labeled composition claim and a stock solution mixture with known concentrations of amitriptyline hydrochloride and perphenazine. They prepare four standards and a pharmaceutical sample of unknown concentration, assay each solution in quadruplicate, and plot average peak areas of the concentrations of the known solutions in the construction of a standard curve. From the mathematical relationships that result, the average masses of amitriptyline hydrochloride and perphenazine in the prescription tablet are determined. Finally, the standard deviations of the mean masses are calculated. The entire laboratory procedure and statistical data analysis can be completed in a single 3-hour period.

Quantitative High-performance Liquid Chromatographic Analysis of Indapamide in Pharmaceutical Dosage Forms

A specific high-performance liquid chromatographic method has been developed for quantitative analysis of indapamide in solid pharmaceutical dosage forms on the Turkish market. A LiChrospher RP-18 column, HP-1050 UV-detector and acetonitrile-distilled water (35:65) as the solvent system were used. The flow-rate was 1.5 mL min−1. Quantitative determination of indapamide was carried out at 254 nm. The method was found to be rapid (under 10min per assay) and was selective enough to allow the determination of indapamide in the presence of certain excipients.

Quantitative HPLC Analysis of an Analgesic/Caffeine Formulation: Determination of Caffeine

A modern high performance liquid chromatography (HPLC) laboratory experiment which entails the separation of acetaminophen, aspirin, and caffeine and the quantitative assay of caffeine in commercial mixtures of these compounds has been developed. Our HPLC protocol resolves these compounds in only three minutes with a straightforward chromatographic apparatus which consists of a C-18 column, an isocratic mobile phase, UV detection at 254 nm, and an integrator; an expensive, sophisticated system is not required. The separation is both repeatable and rapid. Moreover, the experiment can be completed in a single three-hour period. The experiment is appropriate for any chemistry student who has completed a minimum of one year of general chemistry and is ideal for an analytical or instrumental analysis course.The experiment detailed herein involves the determination of caffeine in Goody's Extra Strength Headache Powders, a commercially available medication which contains acetaminophen, aspirin, and caffeine as a...

Quantitative High-performance Liquid Chromatographic Analysis of Famotidine in Pharmaceutical Dosage Forms

A rapid and specific high-performance liquid chromatographic (HPLC) method has been developed for quantitative analysis of famotidine in pharmaceutical dosage forms. The method was applied to medicines on the Turkish market. A LiChrospher RP-18 column, HP-1050 UV-detector and methanol—0·1 M aqueous ammonium acetate (30 : 70) as the solvent system (flow rate 1·0 mL min−1) were used. Quantitative determination of famotidine was carried out at 254 nm wavelength. The method was found to be rapid—under 5 min per assay—and was selective enough to allow the determination of famotidine in the presence of certain excipients.