C18 High-performance Liquid Chromatography Column Research Articles

Characterization of Escherichia coli MoeB and Its Involvement in the Activation of Molybdopterin Synthase for the Biosynthesis of the Molybdenum Cofactor

Amino acid sequence comparisons of Escherichia coli MoeB suggested that the MoeB-dependent formation of a C-terminal thiocarboxylate on the MoaD subunit of molybdopterin synthase might resemble the ubiquitin-activating step in the ubiquitin-targeted degradation of proteins in eukaryotes. To determine the exact role of MoeB in molybdopterin biosynthesis, the protein was purified after homologous overexpression. Using purified proteins, we have demonstrated the ATP-dependent formation of a complex of MoeB and MoaD adenylate that is stable to gel filtration. Mass spectrometry of the complex revealed a peak of a molecular mass of 9,073 Da, the expected mass of MoaD adenylate. However, unlike the ubiquitin activation reaction, the formation of a thioester intermediate between MoeB and MoaD could not be observed. There was also no evidence for a MoeB-bound sulfur during the sulfuration of MoaD. Amino acid substitutions were generated in every cysteine residue in MoeB. All of these exhibited activity comparable to the wild type, with the exception of mutations in cysteine residues located in putative Zn-binding motifs. For these cysteines, loss of activity correlated with loss of metal binding.

Assay of gamma-glutamylcysteine synthetase activity in Plasmodium berghei by liquid chromatography with electrochemical detection.

This work describes a high-performance liquid chromatography (HPLC) method to determine gamma-glutamylcysteine (gamma-GC), the intermediate product of glutathione biosynthesis. Separation relies on isocratic reversed-phase chromatography using a Symmetry C18 HPLC column, particle size 5 microm, 4.6 x 250 mm i.d. The mobile phase is methanol-dibasic sodium phosphate (pH 6.6; 2.8 mM) (10:90, v/v) at the flow-rate of 0.5 ml/min and detection is operated electrochemically (+200 and +550 mV) with a pre-column derivatisation reaction using ortho-phthalaldehyde (OPA) as reagent. Under these conditions the calibration range of gamma-GC was 0.3-10 microg/ml; the limit of quantification was 0.3 microg/ml; accuracy, expressed as %Bias, was <10 and precision (%CV) was <6. The proposed HPLC assay was used to quantitate the gamma-glutamylcysteine produced by the gamma-glutamylcysteine synthetase of the rodent malaria parasite Plasmodium berghei in an in vitro enzymatic assay.

Determination of LY295501 in human plasma by reverse phase HPLC with UV detection

A high performance liquid chromatographic (HPLC) assay was developed and validated for the quantitative determination of LY295501 in human plasma. A structural analog, LY186641, was selected as the internal standard. The samples were processed by protein precipitation with acetonitrile followed by concentration of the supernatants and reconstitution. Chromatographic resolution of LY295501 from endogenous plasma components was accomplished with a Waters Novapak C18 HPLC column (3.9×150 mm, d p 4 mm). Detection was by absorbance at 260 nm. The linear dynamic range was from 5 to 400 μg ml −1 of human plasma using a 0.25 ml aliquot. The inter-day precision (%RSD) and accuracy (%RE) in plasma ranged from 2.4 to 4.7, and −4.9 to 1.4, respectively. This assay is both simple and rapid, and has been used to successfully analyze over 1500 samples from human clinical trials.

Rapid analysis of oxidized cholesterol derivatives by high‐performance liquid chromatography combined with diode‐array ultraviolet and evaporative laser light‐scattering detection

AbstractExtensive evidence of the deleterious biological effects of oxidized 5‐cholesten‐3β‐ol (cholesterol) derivatives has led to great interest in their detection. We observed that known oxidized cholesterol derivatives can be rapidly quantitated by combining reversed‐phase high‐performance liquid chromatography (HPLC) with ultraviolet (UV) absorption and evaporative laser light‐scattering (ELSD) detection. Using a 20 × 0.46 cm C18 HPLC column and methanol/acetonitrile (60:40, vol/vol) as the mobile phase at 1.0 mL/min, 10 species of oxidized cholesterol derivative were measured by UV (205, 234, and 280 nm) while 5‐cholestan‐5α,6α‐epoxy‐3β‐ol (5α‐epoxycholesterol), 5‐cholestan‐5β,6β‐epoxy‐3β‐ol (5β‐epoxycholesterol), and 5‐cholestan‐3β,5α,6β‐triol (cholestanetriol) were detected by only ELSD. The minimal limits of detection ranged from 100 to 500 ng depending on sterol and detector. The response was linear in the range 0–1000 or 0–2000 ng depending on detector. These oxidized cholesterol derivatives were also identified by HPLC/mass spectrometry analysis combined with UV detector. Heated tallow contained cholestanetriol, 5‐cholesten‐3β,7α‐diol (7α‐hydroxycholesterol), 5‐cholesten‐3β,7β‐diol (7β‐hydroxycholesterol), 5‐cholesten‐3β‐ol‐7‐one (7‐ketocholesterol), 5α‐ and 5β‐epoxycholesterols under the developed analysis condition. Photooxidized cholesterol had cholestanetriol, 7α‐ and 7β‐hydroxycholesterols and 3,5‐cholestadien‐7‐one. On the other hand, 7α‐ and 7β‐hydroxycholesterols, 7‐ketocholesterol, 5α‐ and 5β‐epoxycholesterols and 3,5‐cholestadien‐7‐one were observed in copper‐oxidized low‐density lipoprotein. Thus, this developed HPLC analysis method could be applied to identification of oxidized cholesterol derivatives in food and biological specimen.

Improved High-Performance Liquid Chromatographic Method for the Analysis of Potato (Solanum tuberosum) Glycoalkaloids

An improved high-performance liquid chromatographic (HPLC) method for the determination of the potato glycoalkaloids, α-solanine and α-chaconine, is described. The glycoalkaloids were extracted with aqueous acetic acid, concentrated, and cleaned up by solid phase extraction using C18 cartridge columns and separated using a reverse phase C18 HPLC column. Recoveries averaged 93% ± 1.3 for α-solanine and 99% ± 3.1 for α-chaconine. Comparisons were made at each step with existing published methods. We conclude that trifunctional endcapped C18 SPE cartridges were more reliable than traditional monofunctional non-endcapped cartridges and that low carbon load C18 HPLC columns provided an improved separation of glycoalkaloids when compared to columns with a high carbon loading. An acidic extraction medium excluding organic solvents is recommended. Keywords: α-Chaconine; glycoalkaloids; HPLC; potato; α-solanine; Solanum tuberosum

Lipid components that reduce protein solubility of soy protein isolates

AbstractA lipid fraction from a commercial soy protein isolate (SPI), previously found to be detrimental to SPI solubility, was analyzed by size‐exclusion liquid chromatography, by high‐performance liquid chromatography (HPLC), and for chemical composition. The molecular weight of most of this material was greater than 1,100 daltons. This lipid fraction was water‐soluble yet required a strong nonpolar solvent mixture to elute it from a C18 HPLC column. The lipid material was alkaline (pH 8.7) and composed of 3.0% nitrogen, 1.6% phosphorus, 17.5% nonvolatile crude fatty acids primarily hydroxylated), 10.4% long‐chain bases, 9.9% hexuronic acid, 3.2% hexosamine, and 6.6% total sugar. The molecular weight, chemical composition, and physical characteristics (solubility characteristics, surfactant characteristics, and appearance) of this material were all similar to those reported for phytoglycolipid.

Measurement of Nimodipine Metabolism in Rat Liver Microsomes by High-Performance Liquid Chromatography

Abstract An isocratic, rapid, sensitive and selective reversed phase high-performance liquid chromatographic (HPLC) assay with ultraviolet detection has been used to quantify the in vitro nimodipine metabolism in rat liver microsomes. (±) Nimodipine and its metabolites were separated on a C18 HPLC column maintained at 40°C using a mobile phase consisting of ammonium acetate 0.05M pH 6.6 and methanol (40:60 v/v). The within- and between-run coefficients of variation (CVs) were < 4.2 % in the concentration range of 0.5–50 μM. The limit of detection for nimodipine and ist metabolites was in the range of 30 - 80 nM. The formation of nimodipine metabolites may be described by a sigmoid Vmax model according to Hill equation corresponding to enzyme kinetics associated with positive allosteric effect. The assay was accurate, selective and may be used in studies investigating the interaction of drugs, and substances found in daily food e.g. flavonoids, with nimodipine metabolism.

High-performance liquid chromatography with light-scattering detection and desorption chemical-ionization tandem mass spectrometry of milk fat triacylglycerols.

The utility of reverse-phase high-performance liquid chromatography (HPLC), desorption chemical-ionization mass spectrometry (DCI-MS) and tandem mass spectrometry (MS/MS) for the characterization of triacylglycerols in complex mixtures has been further explored. Triacylglycerols of anhydrous bovine milk fat were separated by using two reverse-phase C18 HPLC columns, and eluents were monitored with an evaporative light-scattering detector. Fifty-eight fractions were resolved and analyzed by positive ion isobutane DCI-MS. The formation of protonated molecules and of major fragments corresponding to the random loss of any one of the constituent fatty acids readily identified acyl carbon numbers and the number of double bonds within each fatty acid. MS/MS was only required when the original mass spectra indicated the presence of more than one triacylglycerol or of impurities in a fraction. Protonated molecules produced by DCI were fragmented using high energy collisional activation, and the resulting ions were detected by MS/MS. Odd-chain triacylglycerols were also readily distinguished using this methodology. The positive ion DCI and MS/MS techniques described here demonstrate the usefulness of this approach for the characterization of triacylglycerols in complex mixtures.

Determination of Piroximone in Human Plasma by High-Performance Liquid Chromatography

A rapid and selective high-performance liquid chromatographic (HPLC) method using solid-phase extraction (SPE) for measuring piroximone in plasma samples has been developed. Human plasma and internal standard were pipetted onto a Bond Elut C18 extraction cartridge conditioned with methanol and water. Impurities and proteins were washed out with water. Piroximone and internal standard were eluted with methanol. After evaporation, the residue was dissolved in the mobile phase and the aliquot injected into the HPLC system. Piroximone and its related compounds were separated on a reversed phase C18 HPLC column maintained at 50 degrees C using a mobile phase consisting of phosphate buffer and methanol. Piroximone-N-oxide, piroximone, and internal standard were detected spectrophotometrically at 340 nm. The extraction recovery for piroximone was 94%. The within- and between-run coefficients of variation were < 3% in the concentration range of 320-5,000 ng/ml and < 17.5% at lower concentrations, e.g., 20 ng/ml. The limit of detection was 10 ng/ml.

The HPLC Determination of Total Thiamin (Vitamin B1) in Foods

A high-performance liquid chromatography (HPLC) method for the routine determination of total thiamin in foods is described. After acid and enzymatic hydrolysis, thiamin was oxidized to thiochrome, and the oxidized fraction was purified and concentrated with solid-phase extraction (SPE). Several commercially available SPE columns were tested. Thiochrome was analyzed with a C18 HPLC column, using a mixture of methanol and 50 mM phosphate buffer (pH 7.0) (30:70) as the mobile phase. Fluorescence detection (ex 366 nm, em 436 nm) was used. The method was verified using the 14C technique. Labeled thiamin, [thiazole-2-14C]thiamin hydrochloride, was added to four different food matrices (cheese, pork, potato, and wheat flour), and the distribution of 14C activity was measured at several stages of the analytical procedure. On the average, 14% of the labeled thiamin was lost in the thiochrome oxidation and in the SPE procedure. About 70% of the residual activity was located in the major peak of the reversed-phase thiochrome chromatogram. The total recovery of the added 14C activity was estimated at 60% in both samples and standards. The distribution of activity was found to be similar in the four food matrices and the standards.

Simultaneous determination of seven divalent metal cations in some anaerobic sealant formulations following solid-phase extraction and separation on a dynamically coated C18 high-performance liquid chromatography column

A method has been developed for the simultaneous determination of eight divalent metal ions based on solid-phase extraction and separation on a dynamically coated C18 reversed-phase high-performance liquid chromatography column. The method has been applied to the determination of these metal ions in some anaerobic sealant formulations, with limits of detection as low as 30 ppb for certain metal ions. It has been shown that this approach is capable of distinguishing between free and complexed metal ions in such matrices.

Direct determination of codeine-6-glucuronide in plasma and urine using solid-phase extraction and high-performance liquid chromatography with fluorescence detection.

A sensitive and selective method was developed for the direct determination of codeine-6-glucuronide in plasma and urine using high-performance liquid chromatography (HPLC) with fluorescence detection. Codeine-6-glucuronide was synthesised and its purity estimated using acid and enzyme hydrolysis. The hydrolysis of codeine-6-glucuronide by beta-glucuronidase was incomplete and urine reduced the extent of hydrolysis. Codeine-6-glucuronide was recovered from plasma using a solid-phase extraction column and separated on a reversed-phase C18 HPLC column. The assay showed good reproducibility and accuracy (within 10%), and standard curves were linear between 32 and 1600 ng/ml in plasma and between 0.32 and 160 micrograms/ml in urine. The assay has been applied to the study of the pharmacokinetics and metabolism of codeine in patients.

Separation and purification of lymphocyte chemotactic factor (LCF) and interleukin 2 produced by human peripheral blood mononuclear cells

Two T-cell chemotactic factors, lymphocyte chemotactic factor (LCF) and interleukin 2 (IL-2), were separated and characterized from culture supernatants of concanavalin A-stimulated human peripheral blood mononuclear cells. LCF was purified approximately 7800-fold to homogeneity from culture supernatant using gel filtration and high-performance liquid chromatography (HPLC). LCF was found to he distinct from both IL-2 and interleukin-1. Sephadex G-100 gel filtration of crude supernatants from concanavalin A-stimulated mononuclear cells showed two molecular weight regions of T lymphocyte chemotactic activity. A 10,000- to 25,000-Da region contained both IL-2 and LCF and a 45,000- to 75,000-Da region contained only a high molecular weight form of LCF. Both high and low molecular weight species of LCF eluted with 40–44% acetonitrile from a reversed-phase C18 HPLC column. IL-2 present only in the low molecular weight region eluted from the C18 column with 65–75% acetonitrile. The migration of T lymphocytes to IL-2 was totally inhibited by anti-interleukin 2 receptor antibody while the response of T cells to LCF was unaffected. LCF eluting off the C18 column was purified to homogeneity by two subsequent cycles of gel filtration HPLC. The resultant protein showed a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular weight of 10,500. The data presented here demonstrate that IL-2 and LCF are distinct lymphocyte chemotactic factors and although they are not readily separable from crude supernatants by molecular sieve chromatography, they can easily be distinguished by reversed-phase HPLC.