Isocratic Reversed-phase High-performance Liquid Chromatography Research Articles

Analysis of pyridine dinucleotides in cultured rat hepatocytes by high-performance liquid chromatography

An isocratic reverse-phase high-performance liquid chromatography method for the separation and quantitation of total pyridine dinucleotides in hepatocyte cultures is described. Cells are extracted with cold 3 m perchloric acid or 0.5 n sodium hydroxide containing 50% ( v/v) ethanol and 35% cesium chloride for the determination of the oxidized or reduced pyridine dinucleotides, respectively. Pyridine dinucleotides in the neutralized extracts were separated on an Excellopak ODS C 18 (4.6 × 150 mm) column with 0.1 m potassium phosphate, pH 6.0, containing 3.75% methanol as the mobile phase. NAD + and NADP + were detected spectrophotometrically at 254 nm. The response was linear from 5 to 4000 pmol with recoveries of NAD + and NADP + of 98 and 101.1%, respectively. NADH and NADPH were monitored fluorometrically by activation at 370 nm and emission in the 400–700 nm range. The reduced pyridine dinucleotides had a linear response from 7.5 to 60 pmol with recoveries of NADH and NADPH of 99.4 and 101.3%, respectively. The coefficients of variation for all of the pyridine dinucleotide standards were less than 3.5%.

Surface-enhanced resonance Raman spectroscopy as an ancillary high-performance liquid chromatography detector for nitrophenol compounds.

In this study, the potential application of surface-enhanced resonance Raman scattering (SERRS) spectroscopy as an off-line secondary detector for HPLC has been evaluated. Four nitrophenol compounds, 2-nitrophenol, 4-nitrophenol, 2,4-dinitrophenol, and 4,6-dinitrocresol were separated by isocratic reverse-phase high-performance liquid chromatography (RP-HPLC) and monitored with a conventional UV detector. Resonance Raman (RR) and SERRS spectroscopy were next used to provide the required specificity for distinguishing the nitrophenol compounds. The SERRS detection limit for both 2-nitrophenol and 4-nitrophenol was calculated to be 14 ppb and that for 2,4-dinitrophenol and 4,6-dinitrocresol was estimated to lie near the parts-per-billion level as well. This detection limit is 2-3 orders of magnitude lower than that obtained by RR spectroscopy.

Synthetic peptide purification by application of linear solvent strength gradient theory

The purification of crude synthetic peptide by isocratic reversed-phase high-performance liquid chromatography (RP-HPLC) requires time to determine suitable chromatographic conditions, which limits the use of isocratic RP-HPLC as a purification procedure for the newer, multiple-peptide synthetic techniques. An empirical or semi-empirical procedure to determine the appropriate chromatographic conditions based on a knowledge of the peptide's sequence or molecular weight would allow direct purification of the crude synthetic peptide without the initial exploratory work. Using linear solvent strength gradient theory, a formula was derived which predicts the optimum gradient time from the number of residues in the sought-after peptide and certain chromatographic parameters. Using a variety of crude synthetic peptides of approximately twenty residues and this gradient time, the mass capacity of various commercially available RP-HPLC columns was determined. Typically, the described procedure was capable of isolating <1 mg of purified product on a 250 mm × 10 mm I.D. column in less than 2 h. While this procedure is compatible with the small samples generated by the multiple-peptide synthetic techniques, the mass capacities are lower than has been reported previously. This is due to the definition of “optimum” in linear solvent strength gradient theory.

Separation and determination of theophylline from paraxanthine in human serum by reversed-phase high-performance liquid chromatography

A sensitive and highly specific method for the determination of theophylline in serum by high-performance liquid chromatography (HPLC) has been developed. Theophylline was completely separated from paraxanthine, a major metabolite of caffeine which has been known to interfere with most isocratic reversed-phase HPLC methods, with a mixture of acetonitrile/tetrahydrofuran/acetate buffer (10 mM, pH 5.0; 5:1:94, v/v %) as the mobile phase using a C 18 bonded reversed-phase column. Neither the other xanthine and uric acid derivatives nor numerous drugs tested were found to interfere. The proposed method was applied to therapeutic monitoring utilizing its excellent precision, reproducibility and high specificity for theophylline.

Rapid Method for Cimetidine and Ranitidine Determination in Human and Rat Plasma by HPLC

Abstract A rapid method for determination of cimetidine and rantidine in 100 ul human or rat plasma by isocratic reverse phase HPLC is described. Ranitidine is used as an internal standard for cimetidine assay and vice versa. The procedure consists of one step extraction of alkalinized plasma containing internal standard with 4% v/v isopropranolol in ethyl acetate by vortex for 1 min. The organic phase is transferred and evaporated under a stream of nitrogen. The residue was redissolved with methonal and subsequently eluted on u-Bondapack C18 column at 225 nm. The mobile phase consisted of 8% v/v acetonitrile in 5.0 mM potassium phosphate buffer at pH 5.1. The sample was completed eluted in 9 min. with a detection limit of 0.05 ug/ml for cimetidine and 0.1 ug/ml for ranitidine.

Determination of Dirithromycin, LY281389 and Other Macrolide Antibiotics by HPLC with Electrochemical Detection

Abstract Sensitive and rapid assays have been developed for the determination of the macrolide antibotics dirithromycin, erythromycylamine, and LY281389 in plasma. The methods utilize dichloromethane extraction of alkalinized plasma and isocratic reverse phase HPLC with electrochemical detection. The lower limit of detection is 10 ng/ml. Calibration curves are linear and highly reproducible over the range of 20–500 ng/ml. Precision of the calibration curves is very good having relative standard deviations of 5% or less over the dynamic range. These methods can be used for other macrolide antibiotics with minor modifications to the mobile phase. The electrochemical response of various macrolides was found to be dependent upon the functionality at C-9 of the macrolide ring.

An Efficient Strategy for Optimization of Isocratic Mobile Phase Conditions for HPLC Separation of a Complex Mixture

Abstract Chromatographic resolution of a complex mixture is often a trial and error process. An efficient strategy for the optimization of previously reported isocratic mobile phase conditions utilizes a factorial design and multivariate regression equations to model the dependence of elution time of each component on chromatographic parameters. Several parameters are varied simultaneously, with extreme and central values to allow interpolation in the modeling. Run order is scrambled to minimize time dependent errors. Reported here is the optimization of the separation of monoamine neurotransmitters and metabolites using isocratic reverse phase HPLC chromatography with serial oxidation and reduction electrochemical detection. The predicted retention times closely follow the experimental retention times for a set of conditions not included in the calculation of the model. The results enable the rational adjustment of parameters to meet subsequent chromatographic needs.

Assessment of neuromedin B polyclonal antibodies as molecular probes in neural tissue

Five unique, high affinity rabbit polyclonal antibodies against neuromedin B were characterized in a radioimmunoassay in terms of the following parameters: pH and type of buffer, ionic strength, and non-ionic detergents in order to optimize immunoglobulin-peptide interaction; specificity using peptides of the bombesin family, in addition to the tachykinin substance P: and affinity to neuromedin B. Optimum conditions included acidic pH (5.25), high ionic strength (> 0.1 M) and absence of non-ionic detergents, which inhibited the assay. Affinities for the 5 antibodies ranged from 10 to 48 fmol neuromedin B with titers from 1 : 1,000 to 1 : 10,000 and the sequence-specificity covered the entire peptide; cross-reactivity towards substance P was negligible. As a model tissue, rat spinal cord was homogenized with 5 different extraction solvents, including acetone, methanol, acid and alkaline conditions, and assayed by each polyclonal antiserum; neuromedin B immunoreactivity levels were highest in acid and alkaline extracts and reflected the specificity of the antibody used. Applying these antisera to rat brain extracts, the posterior pituitary gland contained the highest concentration of immunoreactive equivalents of neuromedin B followed by the anterior pituitary, hypothalamus, and hippocampus. The immunoreactive content in the pituitary and hypothalamus, however, depended on the particular antisera used with significant ( P < 0.01) differences existing between them. Further application of these polyclonal antibodies to a spinal cord extract analyzed by isocratic reverse-phase HPLC conditions also revealed differences in their cross-reactivity with the immunoreactive peptides. These antisera may now be used as molecular probes for the determination of extractable immunoreactive neuromedin B from neural tissue and in situ localization by immunohistochemical techniques.

Procedure for quality control of chemiluminogenic labels

A method is presented to monitor the chemical, chemiluminogenic and immunochemical properties of steroid N-(4-aminobutyl)- N-ethylsoluminol (ABEI) labels. The label preparation was applied to an isocratic reversed-phase HPLC system (with on-line ultraviolet detection) and fractionated into seventy 12-s fractions. In the fractions, the immunochemicla response was determined by radioimmunoassay and the chemiluminescence was determined in a luminometer or with photplate detection. From the results of the three detection techniques, conclusions can be drawn about the quality and purity of the label. The presence and identity of immunochemically or chemiluminogenically reactive decomposition products cn also be monitored. As an example, a preparation of 17β-testoterone-3-carboxymethyloxime/aminobutylethylisoluminol is analyzed. Possibilities for quality control, stability control and preparative purification are discussed.

Evidence for isoaspartyl 1 (deamidated) forms of mouse epidermal growth factor

A variant form of mouse submaxillary gland epidermal growth factor (EGF) was identified by isocratic reversed-phase HPLC of EGF obtained by Bio-Gel P-10 column chromatography (“culture grade”). The variant form was essentially absent in preparations of EGF further purified by chromatography on DEAE-cellulose (“receptor-grade” EGF). The spectral properties and amino acid composition of the variant form (EGF-I) could not be distinguished from those of the intact polypeptide isolated by HPLC (α-EGF). Receptor-binding and mitogenic properties of EGF-I were also equivalent to those of α-EGF. These data suggested that EGF-I was structurally very similar to EGF. However, the very low yield (<4%) obtained by Edman degradation indicated that the N-terminal (Asn 1) of the polypeptide was modified. Isoelectric focusing of EGF-I revealed two major immunoreactive bands: one with a p I equivalent to that of α-EGF (p I 4.6) and another at p I 4.1. Alkaline treatment of α-EGF (0.1 m NH 4OH) yielded peak material by HPLC that coeluted with EGF-I; the alkaline-generated EGF-I yielded bands that also focused at pH 4.6 and 4.1. Ammonium hydroxide treatment of [des-Asn 1]-EGF (β-EGF) did not produce conversion to EGF-I. On the basis of these data, we propose that EGF-I was formed by selective deamidation of the N-terminal Asn of intact EGF. This notion is also supported by liquid secondary ion mass spectrometry, which showed that EGF-I was approximately 1.5 mass units greater than α-EGF. The heterogeneity observed by isoelectric focusing supports previous studies which have shown that, following deamidation of N-terminal asparagine, a β-aspartyl shift can occur, which in the present study might yield succinimido-aspartyl 1-EGF and β-aspartyl 1-EGF. Low yields observed during Edman degradation indicate that negligible amounts occur as the α-aspartyl 1-EGF isomer.

High-Performance Liquid Chromatographic Determination of Progesterone

Abstract A practical, rapid, reliable and isocratic reversed-phase HPLC method is described for the determination of progesterone; a method important for determining in cows (1) early pregnancy, (2) reproductive disorders, and (3) timing of artificial insemination. The method is reproducible with a detection limit of 0.5 ng/peak (400 pg/μl) at 254 nm (0.005 AUFS); down by nearly 10-fold from other methods and using simple 254 nm detection. Radioimmunoassay of all eluting fractions demonstrated specificity.

An off‐line two‐dimensional analytical procedure for determination of polycyclic aromatic hydrocarbons in smoke aerosol

AbstractSmoke aerosol from stoves consists of a wide variety of chemical substances of which a number have toxic properties. To study the impact of aerosol emissions on health and environment reliable analytical procedures must be available for these samples. An off‐line two‐dimensional HPLC method is described for the determination of a number of polycyclic aromatic hydrocarbons (PAH). The method consists of a HPLC clean‐up step yielding distinct fractions on activated silica and followed by analysis of each of the fractions by isocratic reversed phase HPLC. Detection is by UV and fluorescence monitors in series. Combination of the chromatographic data obtained from both the clean‐up and analytical step provides additional qualitative information.

Measurement of ϵ- N-trimethyllysine in human blood plasma and urine

A method for measurement of ϵ- N-trimethyllysine in human blood plasma and urine is described. An internal standard, δ- N-trimethylornithine, was added to plasma and urine specimens and the mixtures were deproteinized and/or hydrolyzed. Preliminary purification of ϵ- N-trimethyllysine and δ- N-trimethylornithine was achieved by sequential cation-exchange-anion-exchange chromatography. Amino acids in the column eluates were derivatized with o-phthalaldehyde and mercaptoethanol, and were separated by isocratic reversed-phase high-performance liquid chromatography in the presence of an ion-pairing reagent. Quantitation was achieved by post-column fluorometry. The limit of detection was 5 pmol of ϵ- N-trimethyllysine injected into the chromatograph. The procedure was suitable for determination of ϵ- N-trimethyllysine in 1 ml of plasma or 0.2–0.4 ml of urine. The method was applied to measurements of ϵ- N-trimethyllysine in plasma and urine of four systemic carnitine deficiency patients and six normal subjects. Plasma ϵ- N-trimethyllysine concentration was significantly lower in systemic carnitine deficiency patients compared to normal individuals, but no significant difference in urinary ϵ- N-trimethyllysine excretion was observed between the two groups.

Rapid high-performance liquid chromatography assay for salivary and serum caffeine following an oral load : An indicator of liver function

A rapid isocratic reversed-phase high-performance liquid chromatography (HPLC) system for the quantitative measurement of serum and salivary caffeine is described. The best separation of caffeine from other methylxanthines was achieved by chromatography on an ODS-Hypersil column using a solvent system of 0.1 M ammonium acetate pH 4.6—acetonitrile (85:15, v/v). The effluent was monitored at 280 nm. Caffeine was extracted from diluted serum and saliva samples (10–500 μl) by adsorption on a small Bond-Elut C 18 cartridge and recovered by elution with methanol. Thermospray HPLC—mass spectrometry conditions were optimized to afford a means of directly identifying caffeine in samples. The positive-ion mass spectrum was characterized by an intense protonated molecular ion, MH +, at m/z 195 and negligible fragmentation. When the mass spectrometer was operated in selected ion monitoring mode, caffeine could be detected in less than 1μl of serum and saliva at a concentration of 1 μg/ml. Caffeine (3.5 mg/kg body wt.) was administered orally to healthy adults, children, and newborn infants, and to patients with liver disease. The clearance rate and half-life were determined as a test of liver function. A prolongation in the elimination of caffeine was observed in patients with liver disease and, although there was some overlap in the values obtained for patients with non-cirrhotic liver desease and healthy persons, the oral caffeine load test may usefully serve as a dynamic assessment of liver function in the serial follow-up of patients with liver disease.

Application of high-performance liquid chromatography to the measurement of cortisol secretion rate

After administration of [ 3H] cortisol, the specific radioactivity of the urinary cortisol metabolites is determined by chemical conversion of the 17-hydroxycorticosteroid conjugates to 17-ketosteroids, extraction with Sep-Pak C 18 cartridges, derivatisation with 2,4-dinitrophenylhydrazine and isocratic reversed-phase high-performance liquid chromatography. This allows on-line measurement of ultraviolet absorbance; fractions are collected and 3H is measured by scintillation counting. Alternatively extraction and chromatography are done after enzymic hydrolysis of the steroid conjugates, and the major metabolites are determined colorimetrically in the fractions. The results from the two methods were consistent with each other and with published data.

Band-spacing in reversed-phase high-performance liquid chromatography as a function of solvent strength: A simple and fast alternative to solvent optimization for method development

Numerous reports have described the use of solvent optimization for isocratic reversed-phase high-performance liquid chromatography method development. Solvent optimization involves the use of different solvents (usually methanol, acetonitrile and tetrahydrofuran) to control band-spacing for maximum resolution of the sample. Here, we examine an alternative approach, based on variation of the concentration of organic solvent in the mobile phase (solvent strength). This procedure is less powerful than classical solvent optimization, but it nevertheless possesses a significant ability to effect changes in band-spacing. It is also much more easily carried out. Many samples do not require solvent optimization, and in these cases, a change in solvent strength may be the more practical approach. The retention data required for solvent-strength optimization are most conveniently collected by using two initial gradient runs. The application of gradient retention data for developing a final isocratic separation is facilitated by the use of commercial software. The advantages and limitations of gradient-retention data for this purpose are examined.

Determination of hydroxyproline in tissue collagen hydrolysate by derivatization and isocratic reversed-phase high-performance liquid chromatography

Determination of hydroxyproline in tissue collagen hydrolysate by derivatization and isocratic reversed-phase high-performance liquid chromatography

Synthesis of [ 11C]SCH 23390 for dopamine D1 receptor studies

The optimal conditions for the synthesis of 11C-labeled SCH 23390 by radio-methylation of its desmethyl precursor, SCH 24518, with [ 11C]iodomethane are described. Isocratic reversed phase HPLC was used for the purification of [ 11C]SCH 23390. The specific activity range in 30 runs was 10–235 Ci/mmol and average radiochemical yield was 72% based on [ 11C]iodomethane. Mean synthesis time was 40–60 min from the end of bombardment. Preliminary animal studies indicate that [ 11C]SCH 23390 would be useful in visualizing D1 receptors in a living brain by positron tomography.

Isolation and analysis of bacterial cobamides by high-performance liquid chromatography

Cyanocobamides were extracted from diverse bacterial species, purified by XAD-4 and neutral aluminum oxide column chromatography, and separated by isocratic reversed-phase high-performance liquid chromatography (HPLC). Retention times are given for seven cobamide types: dicyanocobinamide (factor B), Coα-(α-benzimidazolyl)-Coβ-cyanocobamide, Coα-(5-hydroxybenzimidazolyl)-Coβ-cyanocobamide (factor III), Coα-(5-methoxybenzimidazolyl)-Coβ-cyanocobamide (factor III m), Coα-(5-methylbenzimidazolyl)-Coβ-cyanocobamide, cyanocob(III)alamin (vitamin B-12) and Coα-(naphthimidazolyl)-Coβ-cyanocobamide. Other Coβ-ligandyl-cobamides such as hydroxycobamide and the light-sensitive methyl-, acetyl-, propyl-, and adenosylcobamides were separated by HPLC in a gradient mode. The recovery of total cell cobamide after extraction, purification, and separation was 75–80%. The method was useful in preparative and analytical work. Less than 10 ng cyanocobamide was detectable.

Separation of A-ring iodinated oestrogens by isocratic reversed-phase high-performance liquid chromatography

Separation of A-ring iodinated oestrogens by isocratic reversed-phase high-performance liquid chromatography