Simple High Pressure Liquid Chromatography Research Articles

Sugary solutions

Sugary solutions

Caftaric Acid Isolation from Unripe Grape: A "Green" Alternative for Hydroxycinnamic Acids Recovery.

Phenolic acids represent about one-third of the dietary phenols and are widespread in vegetable and fruits. Several plants belonging to both vegetables and medical herbs have been studied for their hydroxycinnamic acid content. Among them, Echinacea purpurea is preferentially used for caffeic acid-derivatives extraction. The wine industry is a source of by-products that are rich in phenolic compounds. This work demonstrates that unripe grape juice (verjuice) presents a simple high-pressure liquid chromatography (HPLC) profile for hydroxycinnamic acids (HCAs), with a great separation of the caffeic-derived acids and a low content of other phenolic compounds when compared to E. purpurea and other grape by-products. Here it is shown how this allows the recovery of pure hydroxycinnamic acids by a simple and fast method, fast protein liquid chromatography (FPLC). In addition, verjuice can be easily obtained by pressing grape berries and filtering, thus avoiding any extraction step as required for other vegetable sources. Overall, the proposed protocol could strongly reduce the engagement of solvent in industrial phenolic extraction.

Development and Validation of a High-Pressure Liquid Chromatography Method for the Determination of Chemical Purity and Radiochemical Purity of a [68Ga]-Labeled Glu-Urea-Lys(Ahx)-HBED-CC (Positron Emission Tomography) Tracer.

Background: Prostate-specific membrane antigen (PSMA) has gained high attention as a useful biomarker in the imaging evaluation of prostate cancer with positron emission tomography (PET) during recent years. [68Ga]-labeled Glu-urea-Lys(Ahx)-HBED-CC ([68Ga]-PSMA-HBED-CC) is a novel PSMA inhibitor radiotracer which has demonstrated its suitability in detecting prostate cancer. Preparation conditions may influence the quality and in vivo behavior of this tracer, and no standard procedure for the quality control (QC) is available. The aim of this study was to develop a new rapid and simple high-pressure liquid chromatography method of analysis for the routine QCs of [68Ga]-PSMA-HBED-CC to guarantee the high quality of the radiopharmaceutical product before release. Methods: A stepwise approach was used based on the quality by design concept of the International Conference of Harmonisation Q2 (R1) and Q8 (Pharmaceutical Development) guidelines in accordance with the regulations and requirements of European Association of Nuclear Medicine, Society of Nuclear Medicine, International Atomic Energy Agency, World Health Organization, and Italian Association of Nuclear Medicine and Molecular Imaging. The developed analytical test method was validated because a specific monograph in the pharmacopoeia is not available for [68Ga]-PSMA-HBED-CC. Results: The purity and quality of the radiopharmaceutical obtained according to the proposed method resulted high enough to safely administrate it to patients. An excellent linearity was found between 0.8 and 5 μg/mL, with a detection limit of 0.2 μg/mL. Assay imprecision (% CV) was <2%. Conclusions: The developed method to assess the radiochemical and chemical purity of [68Ga]-PSMA-HBED-CC is rapid, accurate, and reproducible, allowing routinely the use of this PET tracer as a diagnostic tool for imaging prostate cancer and also assuring patient safety.

Quantification of plasma reduced glutathione, oxidized glutathione and plasma total glutathione in healthy cats.

Glutathione is an important intracellular tripeptide with multiple functions. Abnormal glutathione metabolism is thought to play an important role in various diseases of cats. However, no data regarding concentration of plasma glutathione are available for domestic cats. This study discusses the development of a rapid, simple high pressure liquid chromatography method for measurement of reduced glutathione (GSH), oxidized glutathione (GSSH) and total glutathione (GSHt) in plasma, for the purpose of establishing baseline data for future studies. The mean concentrations of GSH, GSSH and GSHt were 4.51+/-1 microM; 19.44+/-3.79 microM (expressed as GSH equivalent) and 23.59+/-3.89 microM, respectively. This is the first report of plasma glutathione concentrations in this species.

Validation of a Simple and Rapid HPLC Method for Determination of Metronidazole in Dermatological Formulations

A rapid and simple method using an isocratic high-pressure liquid chromatography (HPLC) and UV detection for the determination of metronidazole in dermatological formulations is presented. Metronidazole samples were extracted with a solution composed of 60% methanol and 40% mobile phase by a procedure that can be completed in less than 10 min. Subsequent separation and quantification was accomplished in less than 20 min using reversed-phase HPLC with isocratic elution with 0.01% trifluoroacetic acid/acetonitrile (85:15%, vol/vol). Validation experiments confirmed the precision and accuracy of the method. When applied to a commercial metronidazole cream and gel formulation, recoveries of 100.4% for cream formulations and 102.3% for gel formulations were obtained. The method should facilitate studies of the formulation compatibility of metronidazole topical formulations with agents that may improve its clinical tolerability for treatment of rosacea.

Determination and Quantification of Phenytoin in Human Plasma by Liquid Chromatography with Electrospray Ionization Tandem Mass Spectrometry

A rapid and simple high pressure liquid chromatography method with mass spectrometry detection was developed and validated for the determination of phenytoin in human plasma. Metaxalone was used as internal standard. The sample preparation involves a rapid and simple procedure based on liquid‐liquid extraction. Analysis was performed in less than 3.0 minutes in isocratic mode on a reversed phase C18 column (5μ; 50 × 4.6 mm) using a mobile phase composed of acetonitrile‐buffer 2 mM ammonium acetate (80:20 v/v), pH of buffer adjusted to 3.4 using formic acid, at 0.4 mL min‐1 flow rate. The calibration curves were linear in the measured range between 101.2 ng mL-1 and 5060.0 ng mL-1. The validated lowest limit of quantification was 101.2 ng mL-1 for phenytoin. The mean relative recovery for drug and Internal standard was found to be 78.33% and 77.04%, respectively. The described method has the advantage of being rapid and easy and it could be applied in therapeutic monitoring of these drugs in human plasma.

Pharmacokinetics of CPU0213, a novel endothelin receptor antagonist, after intravenous administration in mice1

To determine the pharmacokinetics associated with acute toxic doses of CPU0213, a novel endothelin receptor antagonist in mice after a single intravenous administration. Concentrations in serum and the pharmacokinetic parameters of CPU0213 were assayed by high pressure liquid chromatography (HPLC) following a single intravenous bolus of CPU0213 at concentrations of 25, 50, and 100 mg/kg in mice. The intravenous acute toxicity of CPU0213 was also assessed in mice. A simple, sensitive and selective HPLC method was developed for quantitative determination of CPU0213 in mouse serum. The concentration-time data conform to a 2-compartment model after iv administration of CPU0213 at concentrations of 25, 50, 100 mg/kg. The corresponding distribution half-lives (T1/2 alpha ) were 3.6, 4.2, 1.1 min and the elimination half-lives (T1/2beta) were 39.4, 70.3, 61.9 min. There was a linear increase in C0 proportional to dose, and the same as AUC(0-t) and AUC(0-infinity);. AUC(0-t) and AUC(0-infinity) were 4.511, 13.070, 23.666 g x min x L(-1 ) and 4.596, 13.679, 24.115 g x min x L(-1), respectively. The intravenous LD50 was 315.5 mg/kg. First order rate pharmacokinetics were observed for CPU0213 within the range of doses used, and the acute toxicity of CPU0213 is mild.

High-Pressure Liquid Chromatographic Determination of Ascorbic Acid in Cooked Sausages

The purpose of this paper was to study and optimize both extraction and high-pressure liquid chromatography (HPLC)–UV detection procedures to develop a proper method for the determination of ascorbic acid content in cooked sausages. A simple and sensitive reversed-phase HPLC method for the NH2-bonded phase has been described for the determination of ascorbic acid content in cooked sausages. Various extracting agents were tested to solubilize the vitamin, with 5% (wt/vol) metaphosphoric acid giving the best results. Samples were chromatographed with UV detection at 248nm on a 25-cm Spherisorb NH2 cartridge with a 0.4-cm inside diameter with a mixture of 0.02M potassium phosphate buffer solution (pH 3.6) and acetonitrile (40:60, vol/vol) for the mobile phase. The method's precision within a day was 1.2%, and its precision between days was 3.8%. The detection limit was 1.6 mg/100 g. Recovery ranged from 91.4 to 96.2% for ascorbic acid added to meat samples. Twenty samples of six different products were analyzed in duplicate. For the samples analyzed, the mean value for ascorbic acid ranged between 21.555 and 24.899 mg/100 g of fresh weight.

A simple high-pressure liquid chromatography cotinine assay: validation of smoking status in pregnant women.

Smoking during pregnancy is a significant public health issue, and studies of the effectiveness of interventions to reduce maternal smoking require accurate measurement of smoking status. This study addresses some key issues in improvement of the effectiveness and efficiency of chemical validation of smoking status using a simplified high-pressure liquid chromatography urine cotinine method. Urine samples were collected from pregnant women enrolled in a smoking cessation trial and from non-pregnant volunteers exposed to environmental tobacco smoke (ETS). Analysis of ETS samples produced a maximum cotinine of 28 microg/mmol creatinine, which was established as the cut-off point for this method. This method is a relatively fast and inexpensive technique with which to analyse large batches of cotinine samples and can reliably measure smoking status.

High-performance liquid chromatographic analysis of the 2-chloroprocaine metabolite, diethylaminoethanol, in blood and serum

Background and Objectives. 2-Chloroprocaine is rapidly metabolized in the blood to yield 2-chloro-para-aminobenzoic acid (an inactive metabolite) and diethylaminoethanol (DEAE). DEAE possesses local anesthetic activity. The only reported assay for DEAE is a colorimetric method. Methods. Clinical samples of whole blood and serum were obtained from patients receiving stepped intravenous infusions of 3% 2-chloroprocaine. A high pH-dependent liquid-liquid extraction step with diethyl ether was used to eliminate interfering peaks in high-pressure liquid chromatography (HPLC) analysis. Separation and quantitation were performed using HPLC on a polymeric-reversed phase column with a mobile phase consisting of 10% or 20% acetonitrile (for whole blood or serum analysis, respectively) in 50 mm aqueous sodium phosphate buffer, pH = 11.50. The elution order of DEAE and its analogues was tested to interpret the HPLC separation mechanism. Results. Extraction recovery of DEAE from whole blood was 67 ± 13.5%, from serum, 71 ± 12.2%, and from water, 75 ± 2.9%. The high pH value of the mobile phase resulted in sharp, well-resolved peaks with run times of approximately 8 minutes using 20% acetonitrile. The lower limit of detection was 5 ng/mL of DEAE from a 1-mL sample. The elution order of DEAE and its analogues indicated that separation was based on the hydrophobicity of the analytes rather than polar group interactions occurring with silica-based stationary phase. Conclusions. A new, simple and rapid HPLC method for extraction and measurement of DEAE in whole blood or serum samples is reported here.

A Rapid HPLC Method for the Determination of Free Fucose in Urine. A Marker of Malignancy in Children

Abstract The concentration of free fucose in urine has been shown to be elevated in adults with malignancy. Current analytical methods include enzymatic analysis and high pressure liquid chromatography (HPLC) with fluorescence detection. We describe a simple HPLC method using anion exchange chromatography with pulsed electrochemical detection for the determination of free fucose in urine of children with malignancy. Separation of sugars is achieved within 7 minutes. The method is linear to 200 μmol/1 fucose with a detection limit of 0.5 μmol/1 in urine. Mean recovery was 96.6% ± 11.8%. Precision was good with coefficient of variation (CV) of 4.3% (25 μmol/1), 3.7% (50 μmol/1) and 4.9% (100 μmol/1). The median fucose: creatinine ratio in healthy children was 13.6 μmol / mmol (5.5–128.5). Children with malignancy had a median fucose: creatinine ratio of 8.1 μmol/mmol (2.6–66.4) which was significantly lower than the control subjects (p=0.038). The method enables rapid determination of free fucose in urine w...

Clinical Application of a Simple HPLC Method for the Sugar Intestinal Permeability Test

SummaryA new high pressure liquid chromatography (HPLC) method has been used to measure urinary sugar levels for the intestinal permeability test with cellobiose and mannitol (C/M test). Urinary specimens have been prepared by simple filtration through a Millipore membrane. The method is highly sensitive (minimal detectable concentration of urinary sugars = 0.01 mg/ml) and reproducible (coefficient variation between samples = 0.47% for cellobiose and 0.25% for mannitol). By this method a C/M test has been performed in a large series of gastroenterological patients. High values of mean urinary C/M percentage recovery ratio (C/M%) were found in 30 children with active gluten‐sensitive enteropathy compared with controls (0.42 ± 0.66 versus 0.014 ± 0.005). In 44 treated celiacs and 34 children with chronic nonspecific diarrhea the mean C/M%s were 0.027 ± 0.018 and 0.021 ± 0.012, respectively. The results of this study confirm that the C/M test is a valuable investigation in the diagnostic studies of children with chronic diarrhea. The simple HPLC method described for the determination of urinary sugar levels should allow a wider diffusion of this test.

Clinical Application of a Simple HPLC Method for the Sugar Intestinal Permeability Test

A new high pressure liquid chromatography (HPLC) method has been used to measure urinary sugar levels for the intestinal permeability test with cellobiose and mannitol (C/M test). Urinary specimens have been prepared by simple filtration through a Millipore membrane. The method is highly sensitive (minimal detectable concentration of urinary sugars = 0.01 mg/ml) and reproducible (coefficient variation between samples = 0.47% for cellobiose and 0.25% for mannitol). By this method a C/M test has been performed in a large series of gastroenterological patients. High values of mean urinary C/M percentage recovery ratio (C/M%) were found in 30 children with active gluten-sensitive enteropathy compared with controls (0.42 +/- 0.66 versus 0.014 +/- 0.005). In 44 treated celiacs and 34 children with chronic nonspecific diarrhea the mean C/M%s were 0.027 +/- 0.018 and 0.021 +/- 0.012, respectively. The results of this study confirm that the C/M test is a valuable investigation in the diagnostic studies of children with chronic diarrhea. The simple HPLC method described for the determination of urinary sugar levels should allow a wider diffusion of this test.

Stability of Ketoconazole in Ethanolic Solutions

AbstractThe stability of the antifungal drug Ketoconazole (Kz) in an ethanolic solution was examined. The ethanolic solutions of Kz were placed in either clear or amber glass bottles and stored either at room temperature or at 8 C for 29 days. Kz was assayed using, a sensitive simple high pressure liquid chromatography procedure. Results demonstrated no statistically significant difference between or among the test solutions over time, regardless of the storage conditions or the concentrations (P>0.05).

A non-invasive method for the study of hepatic drug metabolism in rodents: Antineoplastic drug effects on antipyrine metabolism in mice

A rapid, sensitive and simple high pressure liquid chromatography (HPLC) method is described for the direct analysis of antipyrine in saliva. The detection limit was found to be 1.0 ng/ml of sample, lower than any previously reported method. Accuracy and precision were maintained with as little as 0.5 μl of saliva. Thus the rate of elimination of antipyrine has been monitored non-invasively in rats and for the first time in mice. The antipyrine half-life was found to be 28.9 ± 4.0 (S.E.M.) min and 111 ± 20 min in mice and rats, respectively. In mice single i.p. doses of 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) (30 mg/kg) produced increases in antipyrine half-life, up to 28 days post-treatment. The maximum effect of BCNU was observed on day 7 with an antipyrine half-life of 74.4 ± 15.7 min. Phenobarbital induction lowered the antipyrine half-life in controls to 12.6 ± 1.2 min. An enhanced inductive effect was observed in BCNU-treated mice: BCNU-treated, phenobarbital-induced mice displayed a half-life for antipyrine of 7.4 ± 0.6 min on day 21 post BCNU dose. These effects could not be attributed to changes in absorption of antipyrine in BCNU-treated mice.

Single column high pressure liquid chromatographic determination of drugs in blood

1. Analysis of anticonvulsants (phenobarbital, diphenylhydantoin and carbamazepine), theophylline and an antiarrhythmic agent (disopyramide) in blood using a simple high pressure liquid chromatography apparatus equipped with a reversed -- phase column is described. A simple extraction of plasma or serum with organic solvent is used to isolate the anticonvulsants and theophylline. Disopyramide is extracted with ether and is further purified by a back extraction into acid. 2. Hexanesulfonic acid -- methanol solutions are used for chromatography of the anticonvulsants and disopyramide while the mobile phase for theophylline is a NH4H2PO4 -- methanol mixture. Chromatographic analysis time for the drugs is approximately 15 minutes. The drugs are monitored by a UV detector at 254 nm except for theophylline which is measured at 280 nm. Quantitation is accomplished by comparison of peak heights with those of internal standards. Quantities of serum or plasma routinely used for analysis are: 200 ul for the anticonvulsants, 100 ul for theophylline and 0.5 ml for disopyramide. Detection limits are less than 1 ug/ml for these quantities.