Validation Method for the Determination of Sulfonamide Residues in Bovine Milk by HPLC

A simple, rapid, sensitive and reliable high-performance liquid chromatographic method for the simultaneous determination of eight sulfonamides (SAs) in bovine milk was developed (sulfadiazine, sulfathiazole, sulfamethazine, sulfamethoxypyridazine, sufamonomethoxine, sulfamethoxazole, sulfadimethoxine and sulfaquinoxaline) in bovine milk was developed. Samples were prepared by extraction with ethyl acetate and cleaning-up with an anion solid-phase extraction (SPE) column. Analytical separation was performed on an Inertsil ODS-3 column with photodiode-array detection at 270 nm under gradient condition. The whole procedure was evaluated according to the European Commission Decision 2002/657/EC. Specificity, decision limit (CCα), detection capacity (CCβ), trueness and precision were determined during the validation process. It was found that the analytes were isolated from spiked samples with good recoveries between 70.5 and 89.0%. The used analytical conditions allow to successively separating all the tested sulfonamides with good limit of detection between 0.8 and 1.5 μg L⁻¹.     

Determination of Eprinomectin in Bovine Urine and Feces Using HPLC with Fluorescence Detection

Eprinomectin is a novel and potent antiparasitic animal health drug. An analytical procedure for the determination of EPR in bovine urine and feces has been developed. The urine sample was centrifuged and alkalized with ammonia following solid phase extraction. The fecal sample was extracted with acetonitrile, defatted with hexane, cleaned-up using C18 cartridge. All samples were analyzed by high performance liquid chromatography with fluorescence detection after derivatization with N-methylimidazole. The limits of detection are 0.5 ng mL⁻¹ and 0.5 ng g⁻¹, respectively. Fortified at 2, 10, 50, and 100 ng mL⁻¹(ng g⁻¹), inter-assay recoveries of EPR in cattle urine and feces were in the range of 87.9–91.5% and 78.6–86.3%, with coefficients of variation of 5.4–10.2% and 1.4–7.2%, respectively. Intra-assay mean recoveries of the analytes were 82.2–86.5% and 79.6–87.3%, with coefficients of variation of 7.8–11.5% and 6.3–7.8%, respectively. The method was used to study the excretion of eprinomectin in bovine urine and feces after subcutaneous administration at a dose of 0.5 mg kg⁻¹.     

HPLC determination of berberine in plasma of patients with ischemic heart failure

Summary Berberine absorption by patients with ischemic heart failure (IHF) after oral administration and the relationship between clinical effect and plasma berberine concentration are studied. Plasma samples were pretreated by chloroform extraction. Berberine was determined on a μBondapak column with acetonitrile-phosphoric acid mobile phase and UV detection. The limit for berberine in plasma was 8 ng mL⁻¹ for an injection volume of 50 μL. Average berberine recovery was 96.5%. Results showed that improvements in symptoms were more significant for patients with plasma berberine concentration>0.1 mg L⁻¹ than for those with<0.1 mg L⁻¹. Plasma berberine monitoring may be helpful in the treatment of patients with IHF.     

Increased sample throughput in HPLC using sample switching

Summary Column switching methods are described which demonstrate an alternative approach to the reduction of analytical time in HPLC. Although most methods use a combination of techniques, it has been possible to group them into major categories involving column cleanup, sample cleanup, multicolumn methods and boxcar chromatography. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Characterisation of pitch by HPLC

Summary A new high performance liquid chromatography method for the characterisation of toluene-soluble fractions of pitches has been developed. Although a chromatographic system typical of size exclusion chromatography was used, results indicate that, for these structurally complex samples, separation does not follow the usual discrimination by molecular size. A differentiation between several classes of polyaromatic hydrocarbons is achieved instead. Data are reported on the analysis of individual standard polyaromatic hydrocarbons, showing that four different elution ranges can be observed: three ofcata-condensed compounds (Cata1, Cata2 and Cata3) and one ofperi-condensed compounds (Peri). Results are reported proving the capacity of this high performance liquid chromatography method to distinguish between pitches of different origin and nature. It is also effective for the study of the chemical reactions occurring during heat treatment.     

HPLC assay of enzymatic activities

Summary Instead of traditional methods, high-performance liquid chromatography can be used for the determination of enzymatic activities. Assays can be performed once the reaction mixture have been injected in the HPLC apparatus. The elution of the samples is carried out with suitable buffers, and the product(s) formed and left oven substrate(s) are quantitatively determined by means of an internal standard. HPLC assay is rapid, selective and comparable with traditional methods. Enzymatic activity can be easily measured in international units and the whole assay performed within 10–20 minutes. The results obtained for the urokinase assay are provided as a detailed example in the text     

HPLC of porphyrinogens with electrochemical detection

Summary A reversed-phase system is described for the separation of coproporphyrinogen I, II, III, IV isomers, deethylisocoproporphyrinogen and isocoproporphyrinogen. The porphyrinogens are detected electrochemically with high sensitivity. The relative retention of the prophyrinogens is mainly governed by the arrangement of the ethyl and methyl substituents around the macrocycle, although steric effect may also be an important parameter.     

HPLC determination of volatile phenols in wines

Summary An alternative to the traditional solvent extraction method used to extract and rapidly quantify ethyl-and vinylphenol and ethyl-and vinylgaiacol from wine is presented. The method is based on retention of volatile phenols on adsorbants. Among the tested resins, the most efficient, AG 2-X8 (anion exchange resin), worked as well with a synthetic solution as with wines. The percolation of clarified wine adjusted to pH 9 on this resin permits, in particular, the elimination of organic acids. Phenols are not eluted after rinsing the column with 1N HCl, but are eluted with methanol after this treatment. Good recovery (91 %) and good repeatability are observed. The eluate is directly analysed by HPLC on an RP18 column after two-fold dilution in water. The four volatile phenols were completely separated and detected by UV at 280 nm with high sensitivity (20–40 ppb). No interference with other compounds were noted in the different wines analysed.     

Modified polystyrene,stationary phases for the separation of different aromatic hydrocarbon types by HPLC

Summary One possible method of characterizing chemicall-modified stationary phases is to describe their chromatographic properties. In this study we investigated several chemically-modifiedpolystyrene-divinylbenzene packings for the separation of nitroarenes, polycyclic aromatic hydrocarbons and aminosubstituted aromatic hydrocarbons. Chromatography was carried out on commercially available polymer stationary phases, for example C-18 or vinylpyridine modified polystyrene. In addition, a chemically-immobilized polymer packing was prepared by introducing nitrogroups, which were established by elemental analysis and infrared spectroscopy.     

HPLC determination of the flavonoid glycosides fromBetulae folium extracts

Summary The main flavonoid glycosides ofBetulae folium extracts (quercetin-3-glucuronide, myricetin-3-galactoside, hyperosid, quercetin-3-arabinoside and quercetin-3-rhamnoside) have been separated by isocratic elution on a C₁₈ Aquapore RP-300 column. Elution was performed with 17% isopropanol at pH 6.2 confirming the validity of this eluent for the analysis of the flavonoid glycosides.     

An HPLC study of tinidazole hydrolysis

Summary The high-performance liquid chromatographic (HPLC) method herein described allows the simultaneous determination of the hydrolysis kinetics of tinidazole and the formation kinetics of the hydrolysis products. Tinidazole is easily hydrolysed under basic conditions at raised temperature. The rate varies with the pH and the temperature of the solution, and the decomposition follows apparent first-order kinetics. The Arrheinius equation can be used to describe the effect of temperature on the half-life.     

Quantitative analysis of mebeverine in dosage forms by HPLC

Summary A simple and rapid, high performance liquid chromatographic (HPLC) procedure for determination of mebeverine in dosage forms (tablet and liquid) is described. Reversed-Phase chromatography was carried out using a mobile phase containing 0.05 M ammonium acetate buffer and acetonitrile, [(45%, v/v) pH 5.2] with UV-detection (263 nm). Replicate regression analyses of three standard plots in the concentration range of 0.5–10 mcg mL⁻¹ obtained on three different days gave a correlation coefficient >0.9995 and the coefficient of variation of the slopes <2.2%. The assay was precise within day and between days as indicated by ANOVA test. The recoveries from 10 replicate tablets of two commercial mebeverine brands and liquid were in order 99.3, 100.5 and 100.1% of the label amount and their coefficient of variations were 1.41, 0.89 and 0.69%, respectively. The limit of quantitation of mebeverine was 5 ng mL⁻¹.     

Simultaneous assay of platelet and plasma catecholamines by HPLC with coulometric detection

Summary Platelets and monoaminergic neurons share many morphological, biochemical, and pharmacological properties. In addition to its similarities to serotonergic nerve endings, the platelet has also been considered a good model for the study of the noradrenergic function, since it can selectively take up, store, and release norepinephrine (NE).In this study platelet and plasma levels of free catecholamines (CA) in 20 healthy subjects have been determined by HPLC with a reversed phase (C₁₈) ionpairing system equipped with a coulometric detector. Direct correlation was observed between platelet and plasma levels of NE. A positive correlation was also found between the age of the sujects and both platelet and plasma NE levels.While showing that the peripheral noradrenergic hyperactivity of the elderly is also reflected in platelet NE content, this study also demonstrates that simultaneous assay of platelet and plasma CAs can be a useful tool for an integrated and more complete evaluation of sympathetic nervous system activity.     

Separation of urea and other polar compounds by HPLC

Summary Urea, glycolic and aminoxyacetic acid amides are the polar metabolites of 2-acetyl-3-phenyl-tetrahydro-1,2,4-oxadiazin-5-one (RGH 4615). They cannot be separated on octadecyl-, cyanosilyl- or amino-phase columns, but silica, used with C₃-C₄ alcohol and water mixtures as the eluent permits their separation. Besides refractive index detection and on-line radioactivity measurement the metabolic formation of¹⁴C-urea was proved by enzymatic cleavage into¹⁴CO₂.     

Quantitative determination of lansoprazole in human serum by HPLC

Summary Lansoprazole is a new inhibitor of gastric proton secretion. An HPLC method for the quantitative determination of lansoprazole in serum is described. The method consists of liquid-liquid extraction and enrichment of the analyte and subsequent reverse-phase liquid chromatography with UV detection. The method is specific, sensitive and practical. It has been applied to serum from healthy volunteers. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

An application of fast protein liquid chromatography (FPLC) in the purification of retinol binding protein from rat serum

Summary Retinol Binding Protein (RBP) is the specific plasma protein for the transport of retinol from liver to peripheral tissues. It is a single polypeptide chain of approximately 21 KDa, and circulates as a 11 molar complex with transthyretin (TTR). The relative low concentration in plasma (40–50 g/ml and its chromatographic behaviour on ionic exchangers render the purification of rat RBP particularly laborious. In this paper we report a simple and semi-automatic method for the preparative purification to homogeneity of rat serum RBP. The method includes: (1) Selective removal of albumin by affinity chromatography on a Blue Sepharose column; (2) Chromatography on a Mono Q strong anion exchange column; (3) Dissociation of the RBP-TTR complex by 3 M urea; (4) Concentration, desalting and freeze drying. The purified RBP has been used for the production in rabbit of antirat RBP specific antibodies for studies on nutritional control of RBP synthesis and metabolism.     

HPLC determination of plasma taurine

Summary The taurine content in plasma was determined by high performance liquid chromatographic analysis of its dansyl derivative with fluorometric detection. After the reaction with dansyl chloride, the derivative was extracted from an aqueous mixture by using tetrabutylammonium as a counter-ion. The influences of different tetrabutylammonium salts and of the eluent mixture composition were studied. Omotaurine, added as the internal standard to the plasma samples, assured good reproducibility. The procedure was applied to the determination of taurine in specimens from different mammalian species.     

A photometric flow measurements method for characterisation of HPLC pumps

Summary The measurement of flow constancy and pulsation amplitudes of HPLC pump based on the photoconversion of malachite green leucocyanide is described. The irradiation time and hence the degree of conversion of the leucocyanide is correlated to flow fluctuations of piston driven HPLC pumps. It is possible to measure the amplitude of pulsations and determine the constancy of the flow rate. Most of the pumps show a flow stability better than ±1% measured as relative standard deviation of the flow rate under HPLC conditions (pressure drop 100 bar at 1 ml/min flow rate). The most expensive pump of those tested showed less than ±0.5% flow instability, however, this result was achieved by installation of a large-volume pulsation damper. The method described also allows flow rate measurements under FIA conditions where there is little or no pressure drop at the pump outlet.     

HPLC with Diode-Array Detection for Determination of Leflunomide in Tablets

We have developed and validated a simple HPLC method for analysis of leflunomide in tablets. Method conditions were determined by assay of a photodegraded sample of leflunomide. Optimum chromatographic performance was obtained with a C₁₈ column and acetonitrile-water as mobile phase. Comparison of spectra recorded with a diode-array detector during elution of the leflunomide peak enabled determination of method specificity. The method is highly sensitive (detection limit 10 ng mL⁻¹) and robust to deliberate variation of the conditions (RSD of peak area < 2.0%). Precision and accuracy were adequate over the concentration range 10 to 100 μg mL⁻¹. These results show the proposed method is suitable for its intended use.     

Determination of grepafloxacin in plasma samples by HPLC: Application to clinical pharmacokinetic studies

Summary A sensitive HPLC assay for the determination of grepafloxacin (GRE) in biological samples is described. Sample preparations were carried out by adding phosphate buffer (pH 7.4, 0.1M), followed by extraction with trichloromethane. GRE and the internal standard, enrofloxacin (ENR), were separated on a reversed-phase column using an aqueous phosphate solution-acetonitrile (78∶22) mobile phase. The concentrations of ENR and GRE eluting of the column with retention times of 2.55, and 4.90 min, respectively were monitored by fluorescence atλ _ex 338 andλ _em 425 nm. The method was shown to be linear from 5 to 4000 ng mL⁻¹. The detection and quantitation limits were 5 and 10 ng mL⁻¹, respectively. Mean recovery was determined as 90%. Inter- and intra-assay precisions were 3.0% and 3.5% respectively. The method was applied to the determination of GRE in plasma samples collected during clinical pharmacokinetic studies.