On-line High-performance Liquid Chromatography Research Articles

Programmed temperature vaporizing injector to filter off disturbing high boiling and involatile material for on-line high performance liquid chromatography gas chromatography with on-column transfer

Insertion of a programmed temperature vaporizing (PTV) injector under conditions of concurrent solvent recondensation (CSR) into the on-line HPLC–GC interface for on-column transfer (such as the retention gap technique with partially concurrent eluent evaporation) enables filtering off high boiling or involatile sample constituents by a desorption temperature adjusted to the required cut-off. Details of this technique were investigated and optimized. Memory effects, observed when transferred liquid was sucked backwards between the transfer line and the wall of the injector liner, can be kept low by a small purge flow rate through the transfer line at the end of the transfer and the release of the liquid through a narrow bore capillary kept away from the liner wall. The column entrance should be within the well heated zone of the injector to prevent losses of solute material retained on the liner wall during the splitless period. The desorption temperature must be maintained until an elevated oven temperature is reached to prevent peak broadening resulting of a cool inlet section in the bottom part of the injector.

Enzyme-based online monitoring and measurement of antioxidant activity using an optical oxygen sensor coupled to an HPLC system

It is estimated that up to 50% of the adult population take antioxidant products on a daily basis to promote their health status. Strangely, despite the well-recognized importance of antioxidants, currently there is no international standard index for labeling owing to the lack of standardized methods for antioxidant measurement in complex products. Here, an online high-performance liquid chromatography (HPLC)-based method to detect and measure the total antioxidant capacity of antioxidant samples is presented. In this approach, complex samples containing antioxidants are separated by the HPLC system, which is further coupled to an antioxidant measuring system consisting of an optical oxygen sensor, laccase, and tetramethoxy azobismethylene quinone (TMAMQ). The antioxidants, separated via HPLC, reduce TMAMQ to syringaldazine, which is then reoxidized by laccase while simultaneously consuming O(2). The amount of consumed oxygen is directly proportional to the concentration of antioxidants and is measured by the optical oxygen sensor. The sensor is fabricated by coating a glass capillary with an oxygen-sensitive thin layer made of platinum(II) meso-tetra(4-fluorophenyl)tetrabenzoporphyrin and polystyrene, which makes real-time analysis possible (t(90) = 1.1 s in solution). Four selected antioxidants (3 mM), namely, catechin, ferulic acid, naringenin (used as a control), and Trolox, representing flavonol, hydrocinnamic acid, flavanone, and vitamin E, respectively, were injected into the online antioxidant monitoring system, separated, and then mixed with the TMAMQ/laccase solution, which resulted in oxygen consumption. This study shows that, with the use of such a system, the antioxidant activity of individual antioxidant molecules in a sample and their contribution to the total antioxidant activity of the sample can be correctly assigned.

Dereplication of Phenolic Derivatives ofQualea grandiflora and Qualea cordata(Vochysiaceae) using Liquid Chromatography coupled with ESI-QToF-MS/MS

A rational and selective method using on-line high-performance liquid chromatography (HPLC) coupled with electrospray quadrupole time-of-flight tandem mass spectrometry (ESI-QToF-MS/MS) was established for the dereplication of phenolic derivatives from Qualea grandiflora and Qualea cordata. The selection of the extracts was based on the antioxidant capacity measured by in vitro DPPH assay. The HPLC-ESI-QToF-MS/MS analysis was conducted by on-flow detection, using high-resolution mass/ratio ions as well as collision induced MS/MS experiments for selected protonated ions. The dereplication of the EtOAc fraction from the hydro-alcohol extract from the stem bark of Q. grandiflora allowed the detection of the flavonoids: 3',4',5',5,6,7-hexahydroxy-8-methylflavanone, 8-methyl-naringenine and 3',7-dimethoxy-8-methyl-4',5,7-trihydroxyflavanone, as well as a benzophenone derivatives: bis(4,6-dimethoxy-2-hydroxy-3-methylphenyl)-metanone, 3',4'-dimethoxy-8-methyl-5,6,7-trihydroxyflavanone, 7-methoxy-6-methyl-3',4',5-trihydroxyflavanone, 6,8-dimethyl-3'-methoxy-4',5,7-trihydroxyflavanone and 3',5'-dimethoxy-6,8-dimethyl-4',5,7-trihydroxyflavanone were detected in the EtOAc fraction from the hydro-alcohol extract from the leaves of Q. cordata.

A simple method for methylmercury, inorganic mercury and ethylmercury determination in plasma samples by high performance liquid chromatography–cold-vapor-inductively coupled plasma mass spectrometry

A simple and sensitive method with a fast sample preparation procedure is proposed for the determination of mercury species in plasma/serum. The method combines online high-performance liquid chromatography separation, Hg cold-vapor formation and inductively coupled plasma mass spectrometry detection. Prior to analysis, plasma (250μL) was accurately pipetted into 15mL conical tubes. Then, an extractant solution containing mercaptoethanol, L-cysteine and HCl was added to the samples following sonication for 10min. Quantitative mercury extraction was achieved with the proposed procedure. Separation of mercury species was accomplished in less than 8min on a C8 reverse phase column with a mobile phase containing 3% v/v methanol+97% v/v (0.5% v/v 2-mercaptoethanol+0.05% v/v formic acid). The method detection limits were found to be 12ngL−1, 5ngL−1 and 4ngL−1 for inorganic mercury, ethylmercury and methylmercury, respectively. Method accuracy is traceable to Standard Reference Material (SRM) 966 Toxic Metals in Bovine Blood from NIST. Additional validation was provided by the analysis of a secondary reference serum sample from the INSQ-Canada. Finally, the method was successfully applied for the speciation of mercury in plasma samples collected from volunteers exposed to methylmercury through fish consumption. For the first time to our knowledge, levels of different species of Hg in plasma samples from riverside populations exposed to MeHg were determined.

Development of a manual method for the determination of mineral oil in foods and paperboard

So far the majority of the measurements of mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH) were obtained from on-line high performance liquid chromatography–gas chromatography–flame ionization detection (on-line HPLC–GC–FID). Since this technique is not available in many laboratories, an alternative method with more easily available tools has been developed. Preseparation on a small conventional liquid chromatographic column was optimized to achieve robust separation between the MOSH and the MOAH, but also to keep out the wax esters from the MOAH fraction. This was achieved by mixing a small portion of silica gel with silver nitrate into highly activated silica gel and by adding toluene into the eluent for the MOAH. Toluene was also added to the MOSH fraction to facilitate reconcentration and to serve as a keeper preventing loss of volatiles during solvent evaporation. A 50μl volume was injected on-column into GC–FID to achieve a detection limit for MOSH and MOAH below 1mg/kg in most foods.

Characterization of Polymethoxylated Flavonoids (PMFs) in the Peels of ‘Shatangju’ Mandarin (Citrus reticulata Blanco) by Online High-Performance Liquid Chromatography Coupled to Photodiode Array Detection and Electrospray Tandem Mass Spectrometry

A sensitive HPLC-DAD-ESI-MS/MS method was established to screen and identify the polymethoxylated flavonoids (PMFs) in the peels of 'Shatangju' mandarin ( Citrus reticulata Blanco). Eight PMF standards, including four polymethoxylated flavones, two polymethoxylated flavanones, and two polymethoxylated chalcones, were first to be analyzed in positive mode by CID-MS/MS. On the basis of the ESI-MS(n) characteristics of PMFs and the results of EIC-MS/MS experiment, 32 PMFs including 24 flavones and 8 flavanones or chalcones were screened from the complex extract of the peels of 'Shatangju' mandarin. Among them, 10 PMFs were hydroxylated polymethoxyflavonoids (OH-PMFs), and the rest were all permethoxylated PMFs. This was the first systematic report of the presence of PMFs in the peels of 'Shatangju' mandarin, especially for polymethoxylated flavanones and chalcones. Meanwhile, the contents of the three main PMFs and total flavonoids in the peels of 'Shatangju' were determined by HPLC and UV spectrophotometry, respectively. The results indicated that the developed analytical method could be employed as an effective technique for the characterization of PMFs.

Risk–benefit evaluation of on-line high-performance liquid chromatography analysis for pooling decisions in large-scale chromatography

In the production of a human therapeutic protein from inclusion bodies, product related impurities of very similar size and charge to the product are created as byproducts of the refold process. Their removal is usually challenging even when using chromatography with high performance resins and elution by shallow linear gradients. Additionally, performing this type of separation for commercial production adds increased complexity. To maximize productivity, columns are loaded so high that product elution profiles are not well separated from the impurities and pooling decisions are challenging. In this paper, conventional UV pooling based on fractionation or predefined absorbance based criteria will be compared to pooling based on fast on-line HPLC analytic. The development and implementation in a GMP process will be shown for a specific challenging separation by hydrophobic interaction chromatography. The different approaches have their unique complexities, timelines, uncertainties, and risks during development and implementation as well as during manufacturing. This study presents a probabilistic framework for quantitative comparison of two processes with unequal variability and uncertainty to evaluate the potential benefits of a PAT technology for its routine use in GMP Bioprocess manufacturing.

Development and validation of a rapid HPLC method for the determination of cefdinir in beagle dog plasma integrated with an automatic on-line solid-phase extraction following protein precipitation in the 96-well plate format

The high-performance liquid chromatography (HPLC) coupled with on-line solid phase extraction (SPE) and ultraviolet (UV) detection was developed for determining cefdinir in beagle dog plasma. After simple pretreatment for plasma with 6% perchloric acid, a volume of 100μL upper layer of the plasma sample was injected into the self-made on-line SPE column. The analytes were retained on the trap column (Lichrospher C18, 4.6mm×37mm, 25μm), and the biological matrix was washed out with the solvent (20mM KH2PO4 adjusted pH 3.0) at flow rate of 2mL/min. By rotation of the switching valve, the target analytes could be eluted from trap column to analytical column in the back-flush mode by the mobile phase (methanol–acetonitrile–20mM KH2PO4 adjusted pH 3.0, 11.25:6.75:82, v/v/v) at flow rate of 1.5mL/min, and then separated on the analytical column (Ultimate™ XB-C18, 4.6mm×50mm, 5μm). The complete cycle of the on-line SPE preconcentration, purification and HPLC separation of the analytes was 4min. The UV detection was performed at 286nm. The calibration curves showed excellent linear relationship (R2=0.9995) over the concentration range of 0.05–50μg/mL. The optimized method showed good performance in terms of specificity, linearity, detection and quantification limits, precision and accuracy. This method was successfully applied to quantify cefdinir in beagle dog plasma to support the pre-clinical pharmacokinetic trial.

An on-line high performance liquid chromatography-crocin bleaching assay for detection of antioxidants

An on-line HPLC (high performance liquid chromatography) method for the rapid screening of individual antioxidants in mixtures was developed using crocin as a substrate (i.e. oxidation probe) and 2,2′-azobis(2-amidinopropane dihydrochloride (AAPH)) in phosphate buffer (pH 7.5) as a radical generator. The polyene structure of crocin and AAPH-derived peroxyl radicals resemble the lipidic substrates and radicals found in true food more closely than the popular, albeit artificial, DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS+ (2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate)) do. After separation by a C18 (octadecyl silica) column and UV (ultraviolet) detection, antioxidative analytes react with peroxyl radicals at 90°C and the inhibition of crocin oxidation (i.e. bleaching) is detected as a positive peak by an absorbance detector at 440nm. The method is simple, uses standard instruments and inexpensive reagents. It can be applied for isocratic HPLC runs using mobile phases containing 10–90% organic solvent in water, weak acids or buffers (pH 3.5–8.5). With baseline correction, gradient runs are also feasible. The radical scavenging activity of several natural antioxidants and a green tea extract was studied. After optimisation of conditions such as reagent concentrations and flows, the limit of detection varied from 0.79 to 7.4ng, depending on the antioxidant.

Development of On-Line High Performance Liquid Chromatography (HPLC)-Biochemical Detection Methods as Tools in the Identification of Bioactives

Biochemical detection (BCD) methods are commonly used to screen plant extracts for specific biological activities in batch assays. Traditionally, bioactives in the most active extracts were identified through time-consuming bio-assay guided fractionation until single active compounds could be isolated. Not only are isolation procedures often tedious, but they could also lead to artifact formation. On-line coupling of BCD assays to high performance liquid chromatography (HPLC) is gaining ground as a high resolution screening technique to overcome problems associated with pre-isolation by measuring the effects of compounds post-column directly after separation. To date, several on-line HPLC-BCD assays, applied to whole plant extracts and mixtures, have been published. In this review the focus will fall on enzyme-based, receptor-based and antioxidant assays.

Levels and speciation of arsenic in the atmosphere in Beijing, China

Arsenic levels and speciation in the total suspended particles (TSPs) were quantitatively determined by high performance liquid chromatography on-line coupled with hydride generation atomic fluorescence spectrometry in Beijing, China from February 2009 to March 2011. The high TSP levels fluctuated between 0.07 and 0.79mgm−3, with a mean level of 0.32±0.17mgm−3. The total arsenic concentrations ranged from 0.03 to 0.31μgm−3 (mean: 0.13±0.06μgm−3) in Beijing‘s air. The concentrations of As(III) and As(V) ranged from 0.73 to 20ngm−3 (mean: 4.7±3.6ngm−3) and from 14 to 2.5×102ngm−3 (mean: 67±35ngm−3), respectively. As levels and speciation demonstrated relative higher levels in spring and autumn and lower values in summer and winter. As(V) accounted for 81–99% of the extractable species in the TSP samples which showed that As(V) was the major fraction of the extractable As. Organoarsenic species, monomethylarsonate (MMA) and dimethylarsinate (DMA) were not found in all samples. Higher values of enrichment factors demonstrated that arsenic in TSP mainly come from anthropogenic sources. High As and its species levels in air and respiratory exposure (0.30–0.84μgd−1) attributed to higher excess cancer risk ((4.2±2.0)×10−4) for people in Beijing.

Determination of Mercury Species by High Performance Liquid Chromatography On-line Coupled with Inductively Coupled Plasma-Mass Spectrometry Based on UV-induced Chemical Vapor Generation Interface

Determination of Mercury Species by High Performance Liquid Chromatography On-line Coupled with Inductively Coupled Plasma-Mass Spectrometry Based on UV-induced Chemical Vapor Generation Interface

Determination of Mercury Species by High Performance Liquid Chromatography On-line Coupled with Inductively Coupled Plasma-Mass Spectrometry Based on UV-induced Chemical Vapor Generation Interface

A method for the determination of inorganic mercury(iHg) and methylmercury(MeHg) was developed by HPLC-ICP-MS with a UV-induced chemical vapor generation interface.The operational conditions of system such as mobile phase of liquid chromatography,formic acid concentration of UV-induced reaction reducing agent,length of UV reaction tube and so on were optimized.Under the optimized conditions,the relative standard deviations(RSD,n=7) of the chromatography peak high for the determination of iHg and MeHg are 0.72% and 0.66%,respectively.The linear range for both the mercury species is between 0.1 and 10 μg/L,while the detection limits are 0.011 and 0.0093 μg/L,respectively.Certified reference material(DORM-2 dogfish muscle) was used to validate this new method.The results agreed well with certified values.Meanwhile,the proposed method has been applied for the determination of iHg and MeHg in wetland water samples,and the recoveries for iHg and MeHg are between 99% and 106%,94% and 110%,respectively.

Identification of new dianthrone glycosides from Polygonum multiflorum Thunb. using high-performance liquid chromatography coupled with LTQ-Orbitrap mass spectrometry detection: a strategy for the rapid detection of new low abundant metabolites from traditional Chinese medicines

A simple, rapid method was developed using on-line high-performance liquid chromatography (HPLC)/electrospray ionization (ESI)-linear ion trap-Orbitrap hybrid mass spectrometry (LTQ-Orbitrap) to analyze bioactive constituents in the extract of Polygonum multiflorum Thunb., a traditional Chinese medicine (TCM). The qualitative analysis was carried out both in the negative and positive mode to acquire accurate mass data in full scan and data dependent MSn scan. Twenty eight new dianthrone glycosides were characterized and their fragmentation behaviors were first exploited by our LC-HR-MSn method. The identification and structure analysis of these chemicals provided essential data for further phytochemical studies of P. multiflorum. Moreover, the results of the present investigations indicated the relevance and usefulness of the combination of HPLC and LTQ-Orbitrap MS to detect and identify new natural products.

Characterization of seventy polymethoxylated flavonoids (PMFs) in the leaves of Murraya paniculata by on-line high-performance liquid chromatography coupled to photodiode array detection and electrospray tandem mass spectrometry

A sensitive HPLC-DAD–ESI-MS/MS method was established to screen and identify the polymethoxylated flavonoids (PMFs) in the leaves of Murraya paniculata (L.) Jack. 16 PMF standards were first to be analyzed in positive mode by the CID-MS/MS. For polymethoxylated flavones, the fragments of [M+H− n × 15] + produced by loss of one or more methyl radicals from the protonated molecule, as well as [M+H−16] +, [M+H−28] +, [M+H−29] +, [M+H−31] +, [M+H−33] +, [M+H−43] +, [M+H−44] +, [M+H−46] + and [M+H−61] + fragment ions were detected, which could be taken as their diagnostic characters. For polymethoxylated flavanones and chalcones, their [M+H] + ions usually underwent RDA cleavage fragmentation of the C-ring prior to the similar loss of diagnostic fragment ions as polymethoxylated flavones, which could be adopted as a shortcut to distinguish them from ordinary flavones rapidly. For the PMF glycosides, the neutral loss of the similar fragments with polymethoxylated flavones from their [aglycone+H] + could be adopted as a simple method to screen them out from complex mixture. Based on these characterizations of PMFs and the results of EIC-MS/MS experiment, 70 PMFs including 45 flavones, 17 flavanones or chalcones and 8 PMFs glycosides were screened out from the complex extract of the leaves of M. paniculata. Among them, 16 compounds were unambiguously identified by comparison with reference substances. The results indicated that the developed analysis method could be employed as a rapid, effective technique for structural characterization of PMFs.

Persistent Exposure to Arsenic via Drinking Water in Rural Bangladesh Despite Major Mitigation Efforts

Elevated arsenic levels in tube-well water in Bangladesh have prompted extensive mitigation projects. We evaluated the effectiveness of long-term mitigation efforts by longitudinally measuring arsenic exposure in pregnant women and their children, the most susceptible population groups. The study was nested in a population-based nutrition intervention in Matlab, Bangladesh. Mother-child pairs (n = 1951) were followed from 2001 to 2003, beginning in early gestation and continuing to 5 years postpartum. We measured arsenic concentrations in urine (U-As) of the 5-year-old children by using high-performance liquid chromatography online with hydride generation and inductively coupled plasma mass spectrometry and compared them with earlier childhood U-As and maternal U-As during pregnancy. Children had elevated U-As at 5 years old (median = 51 μg/L, 5th-95th percentiles = 16-355 μg/L), and U-As distribution was similar to that observed in the mothers during gestation. Children's U-As at 5 years old significantly correlated with their U-As at 1.5 years old and to maternal U-As during early and late gestation. Despite major mitigation efforts, arsenic exposure remains highly elevated in rural Bangladesh. Further mitigation strategies are required and must be rigorously evaluated for long-term efficacy.

Rapid Determination of Mercury Species in Sewage Sludge by High-Performance Liquid Chromatography On-line Coupled with Cold-Vapor Atomic-Fluorescence Spectrometry after Ultrasound-assisted Extraction

So far, no research has been focused on the determination of organomercuries in sewage sludge. Here, an ultrasonic extraction method for the rapid determination of methylmercury (MeHg) and ethylmercury (EtHg) in sewage sludge after ultrasound-assisted extraction is proposed. Using TMA (tetramethylammonium hydroxide) as the extractant with 3.0 g copper powder, ultrasonic extraction for 30 min at 70 °C demonstrated to be highly efficient, and was shown a satisfied extraction efficiency for MeHg and EtHg from sewage sludge samples. Determination of mercury species was carried out by high-performance liquid chromatography on-line coupled with cold-vapor atomic fluorescence spectrometry (HPLC-CV-AFS). The obtained results from quality control samples were excellent. The proposed method was also validated by the application to five real samples. The results showed that the developed methodology was practicable and highly reliable. Due to the high toxicity of organomercuries and huge amounts of sewage sludge discharged every year, people should pay particular attention to pollutions from sewage sludge.

On-line high performance liquid chromatography measurements of extracellular metabolites in an aerobic batch yeast (Saccharomyces cerevisiae) culture

We constructed a bioprocess environment enabling automatic sampling from a bioreactor combined with a compact on-line high performance liquid chromatography (HPLC) unit. This setup allowed us to measure extracellular glucose, ethanol, glycerol, and acetate concentrations automatically at 5 min intervals during the cultivation. This environment also provides mechanical measurement of the optical density (OD) of cells and enables us to collect and store (−35°C) samples for further off-line analyses. Among the available devices, the performance of the sampling-analysis unit is by far the best with regard to speed and number of analytes. Both the sampling and analysis phases are easily controlled by software; thus, providing a unique environment to perform various bioprocess activity tasks, whether they would be cell line screening or optimisation of conditions for growth and productivity. Complex research set-ups can be created and continuous automated measurements empower long-term cultivations with a time series. We provide evidence for the applicability of this environment by performing three comparable batch cultivations with Saccharomyces cerevisiae yeast and show that both the on-line sampling and analysis modes produce reliable data for further use in the monitoring and controlling of bioprocesses. On-line data provided new insight into the dynamics of the diauxic shift during aerobic glucose batch cultivation. When cell growth and carbon dioxide production ceased for the first time during the diauxic shift, acetate accumulation and consumption of the remaining glucose below 0.15 g/L continued to occur for 1 h. At the same time, glycerol and ethanol began to be consumed. Samples were also collected during cultivation for later analysis of intracellular metabolites and to collect more valuable information about metabolism.

On-Line Monitoring of a Continuous Pharmaceutical Process Using Parallel Factor Analysis and Unfolding Multivariate Statistical Process Control Representation

On-line high performance liquid chromatography (HPLC) was used to monitor steady state reactions in three reactors (K = 3) over 48.0 h. Different numbers of chromatograms, with J = 1981 retention time points, were recorded for each of the three reactors. Peaks for each chromatogram were baseline corrected and aligned using correlation optimized warping (COW). To make a complete three-way data set of I = 266 chromatograms a cubic Hermite interpolation was performed. The applied bilinear multivariate statistical process control (MSPC) method included the unfolding PCA and the trilinear technique was PARAFAC. Unfolding in reactor (K) mode was the most informative. D-charts and Q-charts were applied to the data to determine samples which were out of control. Confidence limits were then applied to the D and Q-charts and variables with different behaviours from that encapsulated within the reference data set were located. Both bilinear and trilinear methods were found to be useful for process analysis.

Time-resolved SAXS measurements facilitated by online HPLC buffer exchange

Small-angle X-ray scattering (SAXS) is a powerful technique to structurally characterize biological macromolecules in solution. Heterogeneous solutions are inherently challenging to study. However, since SAXS data from ideal solutions are additive, with careful computational analysis it may be possible to separate contributions from individual species present in solution. Hence, time-resolved SAXS (TR-SAXS) data of processes in development can be analyzed. Many reported TR-SAXS results are initialized by a sudden change in buffer conditions facilitated by rapid mixing combined with either continuous or stopped flow. In this paper a method for obtaining TR-SAXS data from systems where the reaction is triggered by removal of a species is presented. This method is based on fast buffer exchange over a short desalting column facilitated by an online HPLC (high-performance liquid chromatography) connected to the SAXS sample cell. The sample is stopped in the sample cell and the evolving reaction is followed. In this specific system the removal of phenol initiates a self-association process of long-acting insulin analogues. For this experiment, data were collected in time series while varying concentrations. The method can be generally applied to other systems where removal of a species or other changes in experimental conditions trigger aprocess.