Monitoring of impurities using high-performance liquid chromatography with diode-array detection: eigenvalue plots of partially overlapping tailing peaks

The chromatogram of ropinirole in the presence of about 5% of a closely eluting impurity, obtained by HPLC with diode-array detection, was analysed by chemometric procedures. Log eigenvalue plots were used to determine the relative composition of regions of the chromatogram. It is shown that since the peaks exhibit tailing, unusual behaviour is found in the plots. This is verified by performing simulations, in which it is demonstrated that peak asymmetry has a pronounced influence on this chemometric approach. In many cases of liquid chromatographic analysis, asymmetric peak shapes are encountered and methods for peak purity assessment should be re-evaluated in the light of these asymmetries.     

Gradient chromatofocusing high-performance liquid chromatography. I. Practical aspects

In this work, a versatile method for generating linear pH gradients using weak anion-exchange HPLC has been developed, which is termed gradient chromatofocusing high-performance liquid chromatography. This method utilizes a linear external pH gradient generated in the mobile phase entering the column (inlet pH gradient), superimposed on an internally-generated pH gradient within the column (column pH gradient), which results from the buffering action of the ion exchanger on the mobile phase and vice versa. The method shows significant advantages over conventional chromatofocusing, including: decreased expense due to the use of common buffer components, ease of adjusting the slope of the pH gradient produced at the outlet of the column (outlet pH gradient) through the manipulation of the inlet pH gradient and the ability of using high concentration buffers in the mobile phase. Chromatography of fibrinogen degradation products was done using gradient chromatofocusing. Bandwidths comparable to conventional chromatofocusing were obtained in the separation of fibrinogen degradation products.     

Comprehensive two-dimensional high-performance liquid chromatography for the isolation of overexpressed proteins and proteome mapping

A two-dimensional liquid chromatographic system is described here which uses size-exclusion liquid chromatography (SEC) followed by reversed-phase liquid chromatography (RPLC) to separate the mixture of proteins resulting from the lysis of Escherichia coli cells and to isolate the proteins that they produce. The size-exclusion chromatography can be conducted under either denaturing or nondenaturing conditions. Peaks eluting from the first dimension are automatically subjected to reversed-phase chromatography to separate similarly sized proteins on the basis of their various hydrophobicities. The RPLC also serves to desalt the analytes so that they can be detected in the deep ultraviolet region at 215 nm regardless of the SEC mobile phase used. The two-dimensional (2D) chromatograms produced in this manner then strongly resemble the format of stained 2D gels, in that spots are displayed on a X-Y axis and intensity represents quantity of analyte. Following chromatographic separation, the analytes are deposited into six 96-well (576 total) polypropylene microtiter plates via a fraction collector. Interesting fractions are analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) or electrospray mass spectrometry (ESI/MS) depending on sample concentration, which both yield accurate (2 to 0.02%) molecular weight information on intact proteins without any additional sample preparation, electroblotting, destaining, etc. The remaining 97% of a fraction can then be used for other analyses, such Edman sequencing, amino acid analysis, or proteolytic digestion and sequencing by tandem mass spectrometry. This 2D HPLC protein purification and identification system was used to isolate the src homology (SH2) domain of the nonreceptor tyrosine kinase pp60c-src and beta-lactamase, both inserted into E. coli, as well as a number of native proteins comprising a small portion of the E. coli proteome.     

High-performance liquid chromatography of proteins

Incubation of liver microsomes with GDP [14C] mannose leads to the formation of lipid-linked derivatives of [14C] mannose, a dolichol phosphate monosaccharide and dolichol pyrophosphate oligosaccharides. Standard procedures for separating these two types of compounds from each other were found to be deficient in that fractions thought to contain only dolichol pyrophosphate oligosaccharides are contaminated with dolichol phosphate mannose. This paper presents a column chromatographic procedure which conveniently separates the products of an 8 min labeling experiment into two components; dolichol phosphate [14C]mannose and a [14C]-mannose containing oligosaccharide which is also lipid bound. When this oligosaccharide is released from the lipid by hydrolysis and chromatographed on Sephadex G-50 or G-15 it gives a single peak with an indicated molecular weight of 1100. However, when this released oligosaccharide is chromatographed on concanavalin A Sepharose it is resolved into two peaks suggesting that there may be 2 oligosaccharide of approximately the same size but different structures. After brief periods of labeling with GDP [14C]mannose (5 s) an additional oligosaccharide of 3 to 4 sugar residues can be found in the dolichol pyrophosphate oligosaccharides fraction. Incubation of liver microsomes with UDP [14C]glucose or UDP[14C]galactose produces oligosaccharide components containing 7--8 sugar residues. Labeling of microsomes with UDP[14C]acetylglucosamine gives rise to three different components, including a lipid bound oligosaccharide containing 3- 5 sugar residues.     

Analysis of prednisolone in plasma by high-performance liquid chromatography

A sensitive, specific high-performance liquid chromatographic procedure for the determination of prednisolone in plasma is described. The organic solvent extract from plasma is chromatographed on a silica gel column using a mobile phase of 0.2% glacial acetic acid, 6% ethanol, 30% methylene chloride in n-hexane on a high-performance liquid chromatograph fitted with an ultraviolet dector (254 nm). Quantitation of plasma samples containing 25 ng/ml prednisolone is reported. Metabolites and endogenous hydrocortisone do not interfere with prednisolone. The determination of prednisolone concentration in plasma following administration of a 10-mg single oral dose to a human subject is described.     

Synthesis and characterization of new zirconia-based polymeric cation-exchange stationary phases for high-performance liquid chromatography of proteins

Ion-exchange chromatography is a major method used for large-scale protein separations. New zirconia-based polymeric cation-exchange HPLC stationary phases have been developed for protein separations. Two routes were employed for the synthesis. In one method, polyethyleneimine (PEI) was adsorbed onto porous zirconia particles and cross-linked with 1,4-butanediol diglycidyl ether (BUDGE). Succinic anhydride was then reacted with the remaining primary and secondary amine groups on PEI to afford anionic functionalities. The second method utilizes poly(acrylic acid) anhydride as both the crosslinker and the stationary phase. The resulting stationary phases act to separate proteins by a weak cation-exchange mechanism with a slight contribution to retention from hydrophobic interactions. In the presence of 20 mM phosphate buffer, Lewis acid/base interactions between the zirconia support and the proteins, which can significantly broaden the peaks, are sufficiently suppressed. The effects of ionic strength, mobile phase pH, and salt type are discussed. Protein mass recovery and loading capacity for protein separations on these phases have been evaluated. These weak cation-exchange stationary phases exhibit good stability under normal separation conditions for months and are stable in alkaline solution up to pH 10. In contrast to zirconia supports modified with small anionic species, these new phases have no limitation on the type of salt used as the eluent, and they exhibit unique selectivities. Therefore, they offer interesting alternatives for protein separations. To our knowledge, this work represents the first successful example of protein separations using porous zirconia-based polymeric phases under normal chromatographic conditions, which will definitely help make zirconia-based supports more useful for bio-separation.     

Observation of a conformational effect in peptide molecule by reversed-phase high-performance liquid chromatography

RP-HPLC of analogues of oxytocin containing a reduced peptide bond was studied using various columns and buffers. Anomalous behavior of one analogue served as a basis for the discussion of possible conformational consequences of this substitution.     

Determination of sinefungin in rat plasma by high-performance liquid chromatography

This is the 31st article in a continuing series of objectives to direct emergency medicine resident experiences on off-service rotations. Neck and torso trauma accounts for a large portion of injuries, and its management is an essential part of training in emergency medicine. Due to the often life-threatening presentations of trauma victims, resident instruction may be conducted at the bedside in difficult and demanding situations. Therefore, it is essential for residents to have specific goals and objectives to guide their acquisition of knowledge required to make critical decisions for patients with major trauma.     

Quantification of flumequine enantiomers in plasma by high-performance liquid chromatography

A 62-year-old woman with pulmonary hypertension underwent mitral valve re-replacement through right thoracotomy. Severe adhesion occurred to the right lung. During pleural dissection the lung collapsed under single-lung ventilation, rapidly elevated pulmonary vascular resistance caused hemodynamic instability. When pulmonary hypertension is preoperatively present, this approach under single-lung ventilation is not recommended.     

Determination of ivermectin in human plasma by high-performance liquid chromatography

A specific reversed-phase HPLC-assay with sensitive fluorometric detection has been developed to measure the potent new antiparasitic agent ivermectin (CAS 70288-86-7) in human plasma (and urine). The lower limit of the method was 1 ng/ml and the intra-/interassay variability averaged 4.5/6.9%, respectively. The assay was applied for measuring plasma (urine) concentrations of ivermectin upto 56 (72 h) following a single oral dose of 6 and 12 mg. No unchanged or conjugated ivermectin could be detected in urine. Plasma concentrations increased linearly with dose but elimination half-life (12.6/13.4 h) was independent of the administered dose. Thus, the method is applicable for monitoring plasma levels during clinical and pharmacokinetic trials with ivermectin to evaluate its most efficacious dosage regimen.     

Analysis of fluoroquinolones in biological fluids by high-performance liquid chromatography

High-performance liquid chromatographic methods for the analysis of fluoroquinolones in biological fluids are reviewed. In particular, sample preparation and handling procedures, chromatographic conditions, and detection methods are discussed. A summary of published high-performance liquid chromatographic assays for individual fluoroquinolones is included.     

Determination of indomethacin residues in poultry by high-performance liquid chromatography

Linear IgA disease (LAD) is characterized by circulating and tissue-bound IgA antibodies against heterogeneous antigens in the cutaneous basement membrane zone. In most cases the cause is unknown, but a minority of cases has been drug induced. We report a 76-year-old man who developed an acute blistering eruption following high-dose penicillin treatment for pneumococcal septicaemia. Indirect immunofluorescence demonstrated dermal binding IgA antibodies, and Western blotting of serum showed reactivity with a 250 kDa dermal antigen corresponding to collagen VII of anchoring fibrils. Indirect immunoelectron microscopy showed antibody labelling in the lamina densa and sublamina densa zone. This is one of the few cases of drug-induced LAD in which the target antigen profile has been characterized, and the first in which the antigen has been shown to correspond to collagen VII.     

Assay of sulfonylureas in human plasma by high-performance liquid chromatography

A sensitive and specific high-performance liquid chromatographic procedure for the determination of chlorpropamide or tolbutamide in plasma in the presence of their metabolites is described. The ether extract of acidified plasma is redissolved in the mobile phase, 17% acetonitrile in 0.05 M aqueous ammonium formate, and chromatographed on a reverse-phase column on a high-performance liquid chromatograph fitted with a UV absorbance detector. Quantitation of plasma samples containing less than 0.5 mug/ml of chlorpropamide and 5 mug/ml of tolbutamide is reported, using these drugs as mutual internal standards. The retention times of the metabolites are such that they do not interfere in the procedure. The assay method was tested in a human volunteer with both drugs and found suitable for single-dose pharmacokinetic studies.     

Determination of dimethindene in human tears by high-performance liquid chromatography

A high-performance liquid chromatographic method is described for the determination of dimethindene in human tears. The tear samples were diluted in a 0.01 M hydrochloric acid-n-propanol mixture to prevent the irreversible adsorption of dimethindene. The diluted samples were directly injected into the chromatographic system to avoid sample pretreatment. The validation data demonstrate that the method is specific, precise and accurate within the calibration range of 12 to 1000 ng/ml dimethindene free base.     

Analysis of the Morgan-Elson chromogens by high-performance liquid chromatography

The Morgan-Elson method for quantitative N-acetylhexosamine analysis is a two-step procedure comprising alkali treatment of the sugar and subsequent condensation of the resulting chromogens with p-dimethylaminobenzaldehyde (Ehrlich's reagent) to yield a colored product. In the present investigation, the products formed in the first step of the procedure were analyzed by high-performance liquid chromatography (HPLC) on a reversed-phase (C18) column, which was eluted with a water-methanol gradient; the absorbance of the effluent was monitored at 229 nm. The profile generated from alkali-treated N-acetylglucosamine exhibited two major peaks, in a ratio of approximately 2.5:1, which accounted for 94% of the total peak area. A third peak, accounting for 3% of the peak area, was eluted in an intermediate position, and several smaller peaks were also observed. The three predominant components, isolated by preparative HPLC, all gave a purple color on addition of Ehrlich's reagent, indicating that they were Morgan-Elson chromogens. The HPLC profile of alkali-treated N-acetylmannosamine was identical to that of the products generated from N-actylglucosamine, as was expected because of the elimination of the asymmetry at C-2 during formation of the chromogens. N-Acetylgalactosamine yielded two major peaks, which were eluted in the same positions as the two major products formed from N-acetylglucosamine, but the intermediate peak seen in the N-acetylglucosamine pattern was absent. The HPLC procedure allowed detection of as little as approximately 25 ng of N-acetylglucosamine and may therefore be of value as an alternative to the complete Morgan-Elson procedure when only small amounts of sample are available for quantitative analysis.     

Rapid separation of urinary acids by high-performance liquid chromatography

Over a hundred acidic urinary constituents were separated within 30 min by using 5-micron octadecyl-silica columns and gradient elution with increasing acetonitrile concentration in dilute aqueous phosphoric acid solution at 70 degrees. The column effluent was monitored with a UV detector at 280 nm or with a fluorescence detector at 260 nm excitation and 340 nm emission wavelengths. The high sensitivity and speed of analysis, the excellent reproducibility and adequate resolution obtained suggest that this technique may be useful to obtain metabolic profiles in routine clinical work.     

High-performance liquid chromatography of amino acids, peptides and proteins. CXXXIX. Impact of operating parameters in large-scale chromatography of proteins

Large-scale chromatography has been playing an important role in downstream treatment processing in biotechnology. In order to improve the productivity, the throughput of the chromatographic equipment was often increased by increasing the flow-rate and/or by increasing the column sample loading. This paper reports the results of a study on the impact of these and other operating parameters in affinity and ion-exchange chromatographic columns when used for protein purification. A sectional model was developed to predict protein adsorption processes in a packed column. The formulations of this mathematical model are presented in the Appendix. The present study was carried out with computer simulation based on this model and using data obtained from laboratory-scale columns. This model can simulate both the adsorption and washing stages of the protein purification process for both porous and non-porous particles. The effects of changing operating parameters were simulated and contour plots were generated for the easy identification of these effects. It was shown that both flow-rate and column loading can have a considerable impact on the processing rate and the yield of the column. As for the column capacity utilization, the impact of changing flow-rate is not significant at column loading of less than 80% in the test case. It was suggest that the present investigation provides a systematic predictive strategy which will greatly reduce the need for expensive, labour-intensive and time-consuming experimental work during process scale-up.