High performance liquid chromatography hyphenated to inductively coupled plasma mass spectrometry for V and Ni quantification as tetrapyrroles

A method was developed for the determination of V and Ni as tetrapyrroles by High Performance Liquid Chromatography hyphenated to Inductively Coupled Plasma Mass Spectrometry (HPLC-ICP-MS) using reversed phase and elution gradient. Chlorinated solvents and tetrahydrofuran were investigated as regard to separation time and ICP-MS detection efficiencies. The final elution gradient program started from pure methanol to a mixture of 20:80 (v/v) chloroform:methanol. External quantification of V and Ni with inorganic standards by flow injection ICP-MS, used online with HPLC, resulted in 95% of recoveries. The Limits of Detection for V during methanol elution and for Ni during the 20% chloroform gradient elution were evaluated by their minimum detectable concentrations, which were, respectively, 5 μg L^− 1 and 8 μg L^− 1. The methodology was applied to polar and resin fractions separated from a Brazilian crude oil and a sediment extract from an oil-polluted area in the Guanabara Bay, Rio de Janeiro, Brazil. Vanadium as tetrapyrroles represented the totality of V content in the polar fraction, whereas Ni was in different polar forms in the resin and sediment extract.     

Determination of phosphorus using capillary electrophoresis and micro-high-performance liquid chromatography hyphenated with inductively coupled plasma mass spectrometry for the quantification of nucleotides

We performed the quantification of phosphorus in deoxynucleotides using capillary electrophoresis (CE) and micro-HPLC (μHPLC) hyphenated with inductively coupled plasma mass spectrometry (ICP-MS). DNA and its component units have conventionally been determined by photometry; however, more selective and sensitive methods are needed for small biological samples. CE and μHPLC offer the advantages of good separation and small consumption of samples, and ICP-MS is a highly sensitive technique for the determination of a chemical element. Therefore, we have developed an interface device for combining CE and μHPLC with ICP-MS for quantifying nucleotides based on phosphorus content. The interface utilizes 4.5 μL/min for nebulizing and effective introduction of the sample into ICP. The samples of nucleotides and free phosphoric acid were well separated in the CE–ICP-MS measurement, and the calibration curves (1–100 μg/mL) of the nucleotides showed a linear (R² > 0.999) increase in intensity. Similarly, the samples of nucleotides were baseline separated using μHPLC–ICP-MS, and the calibration curves of the nucleotides were linear (R² > 0.998). The detection limits of these species and phosphorus in nucleotides using CE–ICP-MS and μHPLC–ICP-MS were 0.77–6.5 ng/mL and 4.0–6.5 ng/mL, respectively. These values were about one or two orders lower than those in a previous report. The sample volumes of these experiments were calculated to be about 10 nL and 50 nL per analysis. Therefore, these analytical methods have the potential to be useful for the determination of biological samples, such as DNA and RNA molecules.     

Hydrophilic interaction liquid chromatography with indirect ultraviolet detection for the separation and quantification of pyrrolidinium ionic liquid cations

A method of hydrophilic interaction liquid chromatography with indirect ultraviolet detection was developed to determine three pyrrolidinium ionic liquid cations, i.e. N-methyl-N-ethyl pyrrolidinium cation ([MEPy]⁺), N-methyl-N-propyl pyrrolidinium cation ([MPPy]⁺) and N-methyl-N-butyl pyrrolidinium cation ([MBPy]⁺). Chromatographic separation was achieved on a hydrophilic column using imidazolium ionic liquids and organic solvents as the mobile phase. The effects of the background ultraviolet absorption reagents, the imidazolium ionic liquids, detection wavelength, organic solvents, column temperature and the pH value of the mobile phase on the separation and determination of pyrrolidinium cations were investigated and the retention behaviors in hydrophilic interaction chromatography were discussed. The optimized chromatographic conditions were selected. Under the optimal conditions, the detection limits (S/N = 3) for [MEPy]⁺, [MPPy]⁺ and [MBPy]⁺ were 0.59, 0.53 and 0.46 mg/L, respectively. The method has been successfully applied to the determination of the three ionic liquids synthesized in our chemistry laboratory. This research results may improve the analytical method of ionic liquid cations.     

Towards early detection of the hydrolytic degradation of poly(bisphenol A)carbonate by hyphenated liquid chromatography and comprehensive two-dimensional liquid chromatography

The hydrolytic degradation of poly(bisphenol A)carbonate (PC) has been characterized by various liquid chromatography techniques. Size exclusion chromatography (SEC) showed a significant decrease in molecular mass as a result of hydrolytic degradation, while ‘liquid chromatography at critical conditions’ (LC-CC) was very successful for observing differences in functionality due to degradation, i.e. the formation of OH end-groups. To characterize and identify the observed differences semi on-line coupling of liquid chromatography to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and Fourier transform infrared spectroscopy (FT-IR) has proved to be very useful.Comprehensive two-dimensional liquid chromatography (2D-LC) was also applied to study the hydrolytic degradation of poly(bisphenol A)carbonate. LC-CC × SEC showed that the formation of poly(bisphenol A)carbonate with OH end-groups occurred over the whole molecular mass range. This information could not be obtained with the separate liquid chromatographic techniques, thereby illustrating the usefulness of 2D-LC.The main degradation processes of poly(bisphenol A)carbonate under the applied hydrolysis conditions turned out to be disappearance of cyclic PC oligomers, chain scission of PC and (subsequent) formation of OH end-groups. FT-IR, SEC and LC-CC have been used to follow the hydrolytic degradation with time. LC-CC proved to be the most promising technique to detect the degradation of poly(bisphenol A)carbonate at an early stage.     

Comprehensive two-dimensional liquid chromatography with on-line Fourier-transform-infrared-spectroscopy detection for the characterization of copolymers

The on-line coupling of comprehensive two-dimensional liquid chromatography (liquid chromatography x size-exclusion chromatography, LC x SEC) and infrared (IR) spectroscopy has been realized by means of an IR flow cell. The system has been assessed by the functional-group analysis of a series of styrene-methylacrylate (SMA) copolymers with varying styrene content. Ultraviolet (UV) detection was used as a detection technique to verify the detection with IR. The LC x SEC-IR functional-group contour plots (comprehensive chromatograms) obtained for styrene were in agreement with the contour plots constructed from the UV signal. In addition, contour plots can be obtained from non-UV-active groups. One such plot, for the carbonyl-stretching vibration of methylacrylate (MA), is shown. Selective detection of MA proved possible using flow cell IR detection. The combination of the contour plots for styrene and MA allowed a full characterization of the copolymer and it was revealed that the present series of SMA copolymers exhibited homogeneous chemical-composition distributions (CCDs). In addition, commercially available fast-SEC columns have been assessed in this study with respect to their potential to serve as second-dimension separation columns.     

Development of high temperature comprehensive two-dimensional liquid chromatography hyphenated with infrared and light scattering detectors for characterization of chemical composition and molecular weight heterogeneities in polyolefin copolymers

The application of high temperature comprehensive two-dimensional (2D) liquid chromatography for quantitative characterization of chemical composition and molecular weight (MW) heterogeneities in polyolefins is demonstrated in this study by separating a physical blend of isotactic-polypropylene, ethylene-random-propylene copolymer, and high density polyethylene. The first dimension separation is based on adsorption liquid chromatography that fractionates the blend from low to high ethylene content. The second dimension is size-exclusion chromatography connected with light scattering (LS) and infrared (IR) detectors. The IR detector shows desired sensitivity and linearity for monitoring analyte concentrations in the eluent after 2D separations. In addition, the compositions of the analytes are also determined from the ratio of two IR absorbances at the specified wavelength regions, an absorbance for measuring the level of methyl groups in polyolefins and another absorbance for measuring concentration. The LS detector is used to determine absolute molecular weight of the analytes from the ratio of the light scattering signal to the IR concentration signal. The ability to obtain concentration, chemical composition, and MW of polyolefins after 2D separation provides new opportunities to discover structure-property relationships for polyolefins with complex structures/architectures.     

Determination of hydralazine and metabolites in urine by liquid chromatography with electrochemical detection

The cyclic voltammetric behavior of hydralazine and its primary metabolites, the pyruvate and acetone hydrazones, was examined in the positive potential range at both conventional and electrochemically pretreated glassy carbon electrodes. The enhanced oxidations observed at the treated surface were used as the basis of amperometric electrochemical detection of the compounds following reversed-phase liquid chromatography. The detection limits so obtained at +0.75 V vs. Ag/AgCl (1, 3, and 5 ng injected, respectively) were comparable to those previously reported for absorption and fluorescence detection approaches employing derivatization/preconcentration procedures. For liquid chromatography with electrochemical detection, however, direct quantitation of all three species in urine samples was readily accomplished without any chemical derivatization or sample treatment operations other than particulate filtration.     

Determination of 1-nitropyrene metabolites by high-performance liquid chromatography with chemiluminescence detection

Three metabolites of 1-nitropyrene, i.e., 3-hydoxy-1-nitropyrene (3-OHNP), 6-hydoxy-1-nitropyrene (6-OHNP) and 8-hydoxy-1-nitropyrene (8-OHNP), were sensitively and selectively detected by HPLC with peroxyoxalate chemiluminescence (CL) detection. In the system, the three OHNPs were reduced to their corresponding amino derivatives through a reduction column packed with Pt/Rh-coated alumina and then concentrated on a concentrator column. By rotating a switching valve, the analytes were eluted into a separator column (ODS), separated and then detected by the bis(2,4,6-trichlorphenyl)oxalate-H(2)O(2) CL system. The detection limits (signal-to-noise ratio=3) were in the range from 5fmol (6-OHNP and 8-OHNP) to 12fmol (3-OHNP) per injection. To demonstrate the proposed method, it was used to determine three compounds in the incubation mixture of 1-nitropyrene and rat S9 mix.     

The combination of liquid chromatography/tandem mass spectrometry and chip-based infusion for improved screening and characterization of drug metabolites

An approach has been developed for drug metabolism studies of non-radiolabeled compounds using on-line liquid chromatography/tandem mass spectrometry (LC/MS/MS) combined with chip-based infusion following fraction collection. The potential of this approach, which improves the data quality compared with only LC/MS analysis, has been investigated for the analysis of in vitro metabolites of tolcapone and talinolol, two compounds with well-characterized metabolism. The information-dependent LC/MS/MS analysis enables the characterization of the major metabolites while the chip-based infusion is used to obtain good product ion spectra for lower level metabolites, to generate complementary MS information on potential metabolites detected in the LC/MS trace, or to screen for unexpected metabolites. Fractions from the chromatographic analysis are collected in 20 second steps, into a 96-well plate. The fractions of interest can be re-analyzed with chip-based infusion on a variety of mass spectrometers including triple quadrupole linear ion trap (QqLIT or Q TRAP) and QqTOF systems. Acquiring data for several minutes using multi-channel acquisition (MCA), or signal averaging while infusing the fractions at approximately 200 nL/min, permits about a 50 times gain in sensitivity (signal-to-noise) in MS/MS mode. A 5-10 microL sample fraction can be infused for more than 30 min allowing the time to perform various MS experiments such as MS(n), precursor ion or neutral loss scans and accurate mass measurement, all in either positive or negative mode. Through fraction collection and infusion, a significant gain in data quality is obtained along with a time-saving benefit, because the original sample needs neither to be re-analyzed by re-injection nor to be pre-concentrated. Therefore, a novel hydroxylated talinolol metabolite could be characterized with only one injection.     

Biogenic internal standards for duall-label radiochemical detection of metabolites in liquid chromatography

Dual-label radiochemical methods for liquid chromatography are described. Two sets of compounds are used, each set with a different isotopic label. The procedures decrease quantitative uncertainties by as much as 30% for samples in which chemical reactions have altered a test compound in compared biological systems. This advantage is especially useful in toxicologic metabolism studies. Determinations or comparisons of several conjugated and unconjugated metabolites are achieved by extraction and high-performance liquid chromatography based on these procedures. Quantitative procedures that include an internal standard, biologically generated by rats exposed to radiolabeled compounds, are used. By means of these internal standards, metabolite concentrations are compared for test organisms, and results are obtained for metabolites common to both organisms. Effects of impurities, isotope exchange, or chemical kinetics are also evaluated utilizing a modification of the technique.     

Sensitive analysis of blood for amodiaquine and three metabolites by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method using oxidative electrochemical detection has been developed for selective and sensitive quantification of the antimalarial drug amodiaquine and three of its metabolites in the blood of dosed individuals. The method requires only one extraction step and has detection limits of 1 ng/ml for amodiaquine and its metabolites desethylamodiaquine and bisdesethylamodiaquine and 3 ng/ml for 2-hydroxydesethylamodiaquine. Minor modification of the mobile phase preserves the chromatographic separation and allows ultraviolet spectroscopic detection, which, although appreciably less sensitive, permits monitoring of levels of amodiaquine and the three metabolites in blood and urine samples if an electrochemical detector is unavailable. Levels of amodiaquine and the three metabolites were determined for two volunteers undergoing a nine-week chemoprophylactic regimen in connection with travel to a malarious area. Data are included to compare the in vitro antimalarial activities against three strains of Plasmodium falciparum of amodiaquine and the three metabolites considered.     

Identification and determination of flavonoids in astragali radix by high performance liquid chromatography coupled with DAD and ESI-MS detection

A method for the analysis of flavonoids in Astragali Radix by high-performance liquid chromatography (HPLC) combined with photodiode-array detection (DAD) and an electrospray ionization (ESI)--mass spectrometry (MS) was developed. After the samples were extracted with ethanol, the optimum separation conditions for these analytes were achieved using a gradient elution system and a 2.0 x 150 mm Shimadzu VP-ODS column. Eight flavonoids were identified to exist in Astragali Radix based on their characteristic UV data and mass spectra. The concentrations of three major components in this herb--ononin, calycosin and formononetin--were determined by LC/ESI-MS in positive selective ion monitoring (SIM) mode. The calibration curves were linear in the range of 0.9~180.0 μg·mL?1 for ononin, 1.8~360.0 μg·mL?1 for calycosin and 1.4~280 μg·mL?1 for formononetin, respectively. The limits of quantification (LOQ) and detection (LOD) were 0.9 μg· mL?1 and 0.2 μg mL?1 for ononin, 1.8 μg mL?1 and 0.5 μg·mL-1 for calycosin, 1.4 μg mL?1 and 0.5 μg·mL?1 for formononetin, respectively. The standard recoveries were between 95.4~104.7%. The developed method was proven to be useful for the quantitative and qualitative analysis of flavonoid constituents in various resources of Astragali Radix.     

Application of reversed-phase high-performance liquid chromatography for structural characterization of abnormal hemoglobins

The various steps for the structural analysis of a hemoglobin molecule (protein isolation, peptide purification, amino acid composition) can be achieved by the unique use of high-performance liquid chromatography. Reversal-phase high-performance liquid chromatography is of special interest for the identification of neutral variants of hemoglobin.     

Rapid quantification and characterization of soyasaponins by high-performance liquid chromatography coupled with electrospray mass spectrometry

A method using high-performance liquid chromatography (HPLC) with electrospray ionization mass spectrometry (ESI-MS) in the negative mode is presented for the quantification and characterization of different soyasaponins using six authentic soyasaponin standards. This method was successfully applied to the rapid separation of diverse soyasaponins, more than 50, including soyasaponins A in different degrees of acetylation, and soyasaponins B in both their 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP)-conjugated and non-conjugated forms in different samples in one single run for only 30 min. Standard calibration curve was linear over the concentration range of 0.010-1.0 mg/L for each soyasaponin. Within-day and day-to-day relative standard deviations were less than 9.2 and 13.1%, respectively.