Pore structure and column efficiency of silica monoliths in high performance liquid chromatography (HPLC)

Five different methods were critically examined to characterize the pore structure of the silica monoliths. The mesopore characterization was performed using: a) the classical BJH method of nitrogen sorption data, which showed overestimated values in the mesopore distribution and was improved by using the NLDFT method, b) the ISEC method implementing the PPM and PNM models, which were especially developed for monolithic silicas, that contrary to the particulate supports, demonstrate the two inflection points in the ISEC curve, enabling the calculation of pore connectivity, a measure for the mass transfer kinetics in the mesopore network, c) the mercury porosimetry using a new recommended mercury contact angle values. rnThe results of the characterization of mesopores of monolithic silica columns by the three methods indicated that all methods were useful with respect to the pore size distribution by volume, but only the ISEC method with implemented PPM and PNM models gave the average pore size and distribution based on the number average and the pore connectivity values.rnThe characterization of the flow-through pore was performed by two different methods: a) the mercury porosimetry, which was used not only for average flow-through pore value estimation, but also the assessment of entrapment. It was found that the mass transfer from the flow-through pores to mesopores was not hindered in case of small sized flow-through pores with a narrow distribution, b) the liquid penetration where the average flow-through pore values were obtained via existing equations and improved by the additional methods developed according to Hagen-Poiseuille rules. The result was that not the flow-through pore size influences the column bock pressure, but the surface area to volume ratio of silica skeleton is most decisive. Thus the monolith with lowest ratio values will be the most permeable. rnThe flow-through pore characterization results obtained by mercury porosimetry and liquid permeability were compared with the ones from imaging and image analysis. All named methods enable a reliable characterization of the flow-through pore diameters for the monolithic silica columns, but special care should be taken about the chosen theoretical model.rnThe measured pore characterization parameters were then linked with the mass transfer properties of monolithic silica columns. As indicated by the ISEC results, no restrictions in mass transfer resistance were noticed in mesopores due to their high connectivity. The mercury porosimetry results also gave evidence that no restrictions occur for mass transfer from flow-through pores to mesopores in the small scaled silica monoliths with narrow distribution. rnThe prediction of the optimum regimes of the pore structural parameters for the given target parameters in HPLC separations was performed. It was found that a low mass transfer resistance in the mesopore volume is achieved when the nominal diameter of the number average size distribution of the mesopores is appr. an order of magnitude larger that the molecular radius of the analyte. The effective diffusion coefficient of an analyte molecule in the mesopore volume is strongly dependent on the value of the nominal pore diameter of the number averaged pore size distribution. The mesopore size has to be adapted to the molecular size of the analyte, in particular for peptides and proteins. rnThe study on flow-through pores of silica monoliths demonstrated that the surface to volume of the skeletons ratio and external porosity are decisive for the column efficiency. The latter is independent from the flow-through pore diameter. The flow-through pore characteristics by direct and indirect approaches were assessed and theoretical column efficiency curves were derived. The study showed that next to the surface to volume ratio, the total porosity and its distribution of the flow-through pores and mesopores have a substantial effect on the column plate number, especially as the extent of adsorption increases. The column efficiency is increasing with decreasing flow through pore diameter, decreasing with external porosity, and increasing with total porosity. Though this tendency has a limit due to heterogeneity of the studied monolithic samples. We found that the maximum efficiency of the studied monolithic research columns could be reached at a skeleton diameter of ~ 0.5 µm. Furthermore when the intention is to maximize the column efficiency, more homogeneous monoliths should be prepared.rn

Microphytobenthos composition in Heron Reef, Australia, by High Performance Liquid Chromatography (HPLC) (July 2012)

We quantified pigment biomarkers by high performance liquid chromatography (HPLC) to obtain a broad taxonomic classification of microphytobenthos (MPB) (i.e. identification of dominant taxa). Three replicate sediment cores were collected at 0, 50 and 100 m along transects 5-9 in Heron Reef lagoon (n=15) (Fig. 1). Transects 1-4 could not be processed because the means to have the samples analysed by HPLC were not available at the time of field data collection. Cores were stored frozen and scrapes taken from the top of each one and placed in cryovials immersed in dry ice. Samples were sent to the laboratory (CSIRO Marine and Atmospheric Research, Hobart, Australia) where pigments were extracted with 100% acetone during fifteen hours at 4°C after vortex mixing (30 seconds) and sonication (15 minutes). Samples were then centrifuged and filtered prior to the analysis of pigment composition with a Waters - Alliance HPLC system equipped with a photo-diode array detector. Pigments were separated using a Zorbax Eclipse XDB-C8 stainless steel 150 mm x 4.6 mm ID column with 3.5 µm particle size (Agilent Technologies) and a binary gradient system with an elevated column temperature following a modified version of the Van Heukelem and Thomas (2001) method. The separated pigments were detected at 436 nm and identified against standard spectra using Waters Empower software. Standards for HPLC system calibration were obtained from Sigma (USA) and DHI (Denmark).

High-performance liquid chromatography (HPLC) and high-performance liquid chromatography mass spectrometry (HPLC/MS) for the analysis of date rape drugs

The drugs studied in this work have been reportedly used to commit drug-facilitated sexual assault (DFSA), commonly known as "date rape". Detection of the drugs was performed using high-performance liquid chromatography with ultraviolet detection (HPLC/UV) and identified with high performance-liquid chromatography mass spectrometry (HPLC/MS) using selected ion monitoring (SIM). The objective of this study was to develop a single HPLC method for the simultaneous detection, identification and quantitation of these drugs. The following drugs were simultaneously analyzed: Gamma-hydroxybutyrate (GHB), scopolamine, lysergic acid diethylamide, ketamine, flunitrazepam, and diphenhydramine. The results showed increased sensitivity with electrospray (ES) ionization versus atmospheric pressure chemical ionization (APCI) using HPLC/MS. HPLC/ES/MS was approximately six times more sensitive than HPLC/APCI/MS and about fifty times more sensitive than HPLC/UV. A limit of detection (LOD) of 100 ppb was achieved for drug analysis using this method. The average linear regression coefficient of correlation squared (r2) was 0.933 for HPLC/UV and 0.998 for HPLC/ES/MS. The detection limits achieved by this method allowed for the detection of drug dosages used in beverage tampering. This method can be used to screen beverages suspected of drug tampering. The results of this study demonstrated that solid phase microextraction (SPME) did not improve sensitivity as an extraction technique when compared to direct injections of the drug standards.

Development of methodology for the determination of organic acids in must, grape juice and wine by high performance liquid chromatography (HPLC).

2012

Fingerprinting of complex mixtures with the use of high performance liquid chromatography, inductively coupled plasma atomic emission spectroscopy and chemometrics

The molecular and metal profile fingerprints were obtained from a complex substance, Atractylis chinensis DC—a traditional Chinese medicine (TCM), with the use of the high performance liquid chromatography (HPLC) and inductively coupled plasma atomic emission spectroscopy (ICP-AES) techniques. This substance was used in this work as an example of a complex biological material, which has found application as a TCM. Such TCM samples are traditionally processed by the Bran, Cut, Fried and Swill methods, and were collected from five provinces in China. The data matrices obtained from the two types of analysis produced two principal component biplots, which showed that the HPLC fingerprint data were discriminated on the basis of the methods for processing the raw TCM, while the metal analysis grouped according to the geographical origin. When the two data matrices were combined into a one two-way matrix, the resulting biplot showed a clear separation on the basis of the HPLC fingerprints. Importantly, within each different grouping the objects separated according to their geographical origin, and they ranked approximately in the same order in each group. This result suggested that by using such an approach, it is possible to derive improved characterisation of the complex TCM materials on the basis of the two kinds of analytical data. In addition, two supervised pattern recognition methods, K-nearest neighbors (KNNs) method, and linear discriminant analysis (LDA), were successfully applied to the individual data matrices—thus, supporting the PCA approach.

Pharmacokinetics of cimifugin in rat plasma after oral administration of the extract of Saposhnikovia divaricatae root : Determination of cimifugin by high performance liquid chromatography coupled with solid phase extraction

High performance liquid chromatography (HPLC) coupled with the solid phase extraction method was developed for determining cimifugin (a coumarin derivative; one of Saposhnikovia divaricatae's constituents) in rat plasma after oral administration of Saposhnikovia divaricatae extract (SDE), and the pharmacokinetics of cimifugin either in SDE or as a single compound was investigated. The HPLC analysis was performed on a commercially available column (4.6 mm x 200 mm, 5 pm) with the isocratic elution of solvent A (Methanol) and solvent B (Water) (A:B=60:40) and the detection wavelength was set at 250 nm. The calibration curve was linear over the range of 0.100-10.040 microg/mL. The limit of detection was 30 ng/mL. At the rat plasma concentrations of 0.402, 4.016, 10.040 microg/mL, the intra-day precision was 6.21%, 3.98%, and 2.23%; the inter-day precision was 7.59%, 4.26%, and 2.09%, respectively. The absolute recovery was 76.58%, 76.61%, and 77.67%, respectively. When the dosage of SDE was equal to the pure compound calculated by the amount of cimifugin, it was found to have two maximum peaks while the pure compound only showed one peak in the plasma concentration-time curve. The pharmacokinetic characteristics of SDE showed the superiority of the extract and the properties of traditional Chinese medicine.

Collection and determination of nucleotide metabolites in neonatal and adult saliva by high performance liquid chromatography with tandem mass spectrometry

Saliva contains a number of biochemical components which may be useful for diagnosis/monitoring of metabolic disorders, and as markers of cancer or heart disease. Saliva collection is attractive as a non-invasive sampling method for infants and elderly patients. We present a method suitable for saliva collection from neonates. We have applied this technique for the determination of salivary nucleotide metabolites. Saliva was collected from 10 healthy neonates using washed cotton swabs, and directly from 10 adults. Two methods for saliva extraction from oral swabs were evaluated. The analytes were then separated using high performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS). The limits of detection for 14 purine/pyrimidine metabolites were variable, ranging from 0.01 to 1.0 mu M. Recovery of hydrophobic purine/pyrimidine metabolites from cotton tips was consistently high using water/acetonitrile extraction (92.7-111%) compared with water extraction alone. The concentrations of these metabolites were significantly higher in neonatal saliva than in adults. Preliminary ranges for nucleotide metabolites in neonatal and adult saliva are reported. Hypoxanthine and xanthine were grossly raised in neonates (49.3 +/- 25.4; 30.9 +/- 19.5 mu M respectively) compared to adults (4.3 +/- 3.3; 4.6 +/- 4.5 mu M); nucleosides were also markedly raised in neonates. This study focuses on three essential details: contamination of oral swabs during manufacturing and how to overcome this; weighing swabs to accurately measure small saliva volumes; and methods for extracting saliva metabolites of interest from cotton swabs. A method is described for determining nucleotide metabolites using HPLC with photo-diode array or MS/MS. The advantages of utilising saliva are highlighted. Nucleotide metabolites were not simply in equilibrium with plasma, but may be actively secreted into saliva, and this process is more active in neonates than adults. (C) 2013 Elsevier B.V. All rights reserved.

Rapid determination of main constituents of packed juices by reverse phase-high performance liquid chromatography: an insight in to commercial fruit drinks

The present work reports the compositional analysis of thirteen different packed fruit juices using high performance liquid chromatography (HPLC). Vitamin C, organic acids (citric and malic) and sugars (fructose, glucose and sucrose) were separated, analyzed and quantified using different reverse phase methods. A new rapid reverse phase HPLC method was developed for routine analysis of vitamin C in fruit juices. The precision results of the methods showed that the relative standard deviations of the repeatability and reproducibility were < 0.05 and < 0.1 respectively. Correlation coefficient of the calibration models developed was found to be higher than 0.99 in each case. It has been found that the content of Vitamin C was less variable amongst different varieties involved in the study. It is also observed that in comparison to fresh juices, the packed juices contain lesser amounts of vitamin C. Citric acid was found as the major organic acids present in packed juices while maximum portion of sugars was of sucrose. Comparison of the amount of vitamin C, organic acids and sugars in same fruit juice of different commercial brands is also reported.

Analysis of estrogens in environmental waters using polymer monolith in-polyether ether ketone tube solid-phase microextraction combined with high-performance liquid chromatography

A simple, rapid and sensitive on-line method for simultaneous determination of four endocrine disruptors (17 beta-estradiol, estriol, bisphenol A and 17 alpha-ethinylestradiol) in environmental waters was developed by coupling in-tube solid-phase microextraction (SPME) to high-performance liquid chromatography (HPLC) with fluorescence detection (FLD). A poly(acrylamide-vinylpyridine-NAP-methylene bisacrylamide) monolith, synthesized inside a polyether ether ketone (PEEK) tube, was selected as the extraction medium. To achieve optimum extraction performance, several parameters were investigated, including extraction flow-rate, extraction time, and pH value, inorganic salt and organic solvent content of the sample matrix. By simply filtered with nylon membrane filter and adjusting the pH of samples to 6.0 with phosphoric acid, the sample solution then could be directly injected into the device for extraction. Low detection limits (S/N = 3) and quantification limits (S/N = 10) of the proposed method were achieved in the range of 0.006-0.10 ng/mL and 0.02-0.35 ng/mL from spiked lake waters, respectively. The calibration curves of four endocrine disruptors showed good linearity ranging from quantification limits to 50 ng/mL with a linear coefficient R-2 value above 0.9913. Good method reproducibility was also found by intra- and inter-day precisions, yielding the RSDs less than 12 and 9.8%, respectively. Finally, the proposed method was successfully applied to the determination of these compounds in several environmental waters. (c) 2006 Elsevier B.V. All rights reserved.

Artificial neural network classification based on high-performance liquid chromatography of urinary and serum nucleosides for the clinical diagnosis of cancer

Nucleosides in human urine and serum have frequently been studied as a possible biomedical marker for cancer, acquired immune deficiency syndrome (AIDS) and the whole-body turnover of RNAs. Fifteen normal and modified nucleosides were determined in 69 urine and 42 serum samples using high-performance liquid chromatography (HPLC). Artificial neural networks have been used as a powerful pattern recognition tool to distinguish cancer patients from healthy persons. The recognition rate for the training set reached 100%. In the validating set, 95.8 and 92.9% of people were correctly classified into cancer patients and healthy persons when urine and serum were used as the sample for measuring the nucleosides. The results show that the artificial neural network technique is better than principal component analysis for the classification of healthy persons and cancer patients based on nucleoside data. (C) 2002 Elsevier Science B.V. All rights reserved.

Drug-protein interaction studied by technique of microdialysis combined with high performance liquid chromatography

Drug-protein binding is an important process in determining the activity and fate of a pharmaceutical agent once it has entered the body. This review examines the method of microdialysis combined with high-performance liquid chromatography (HPLC) that has been developed;by ours to study such interactions, in which the microdialysis was applied to sample the free drug in the mixed solution of drug with protein, and HPLC to quantify the concentration of free drug in the microdialysate. This technique has successfully been used for determining various types of binding interactions between the low affinity drugs, high affinity drugs and enantiomers to HSA. For the case of competitive binding of two drugs to a protein in solution, a displacement equation has been derived and examined with four nonsteroidal anti-inflammatory drugs and HSA as model drugs and protein, respectively. Microdialysis with HPLC was adopted to determine simultaneously the free solute and displacing agent in drug-protein solutions. The method is able to locate the binding site and determine affinity constants even up to 10(7) L/mol accurately.

Chiral separation of enantiomers of amino acid derivatives by high-performance liquid chromatography on a norvancomycin-bonded chiral stationary phase

A novel norvancomycin-bonded chiral stationary phase (NVC-CSP) was synthesized by using the chiral selector of norvancomycin. The chiral separation of enantiomers of several dansyl-amino acids by high-performance liquid chromatography (HPLC) in the reversed-phase mode is described. The effects of some parameters, such as organic modifier concentration, column temperature, pH and flow rate of the mobile phase, on the retention and enantioselectivity were investigated. The study showed that ionic, as well as hydrophobic interactions were engaged between the analyte and macrocycle in this chromatographic system. Increasing pH of buffers usually improved the chiral resolution for dansyl-alpha-amino-n-butyric acid (Dns-But), dansyl-methionine (Dns-Met) and dansyl-threonine (Dns-Thr), but not for dansyl-glutamic acid (Dns-Glu) which contains two carboxylic groups in its molecular structure. The natural logarithms of selectivity factors (In alpha) of all the investigated compounds depended linearly on the reciprocal of temperature (1/T), most processes of enantioseparation were controlled enthalpically. Interestingly, the process of enantioseparation for dansyl-threonine was enthalpy-controlled at pH of 3.5, while at pH of 7.0, it was entropy-controlled according to thermodynamic parameters Delta(R,S)DeltaHdegrees and Delta(R,S)DeltaSdegrees afforded by Van't Hoff plots. In order to get baseline separation for all the solutes researched, norvancomycin was also used as a chiral mobile phase additive. In combination with the NVC-CSP remarkable increases in enanselectivity were observed for all the compounds, as the result of a "synergistic" effect. (C) 2003 Elsevier B.V. All rights reserved.

Quantitative and qualitative analysis of flavonoids in leaves of Adinandra nitida by high performance liquid chromatography with UV and electrospray ionization tandem mass spectrometry detection

The extract of Adinandra nitida leaves, named as Shiyacha in China, was studied by high performance liquid chromatography (HPLC)-ultraviolet detection-electrospray ionisation (ESI) tandem mass spectrometry (MS). Under the optimized condition, the analysis could be finished in 45 min on a Hypersil C18 column combined with negative ion detection using information-dependent acquisition (IDA) mode of a Q TRAP (TM) instrument. Six flavonoids were identified as epicatechin, rhoifolin, apigenin, quercitrin, camellianin A, and camellianin B among which rhoifolin was for the first time found in Shiyacha. And the fragment pathways of these flavonoids were elucidated. Furthermore, with epicatechin, rhoifolin, and apigenin as markers, the quality control method for Shiyacha and its relevant product was firstly established. Calibration linearity was good (R-2 > 0.9992) over a three to four orders of magnitude concentration range with an S/N = 3 detection limit of 2.5 ng. (c) 2004 Elsevier B.V. All rights reserved.

Analysis of the estrogenic components in kudzu root by bioassay and high performance liquid chromatography

The estrogenic activity of the Chinese herb kudzu root was investigated by a recombinant yeast screening assay (YES). Isoflavones are the main components in the plant, of which puerarin is the most abundant one. The kudzu root extract was separated into four fractions according to the polarity. The crude extract and its sub-fractions, except the water fraction, showed clear estrogenic activity and the potencies were in the range of 10(-3) to 10(-1) g/l. The ligand potency was used to compare the estrogenic activity of these fractions. The crude extract and its sub-fractions were further analyzed by high performance liquid chromatography (HPLC) to correlate the activity and the active components. Bioassay and chemical analysis showed that theoretical estrogenic activity expressed as equivalent 17 beta-estradiol concentration or the cumulative effects are comparable to that experimentally determined by YES. The results showed that the high content of isoflavones as well as the high estrogenic activity could make kudzu root extract an interesting candidate for hormone replacement therapy. (c) 2005 Elsevier Ltd. All rights reserved.