Determination of erythromycins in fermentation broth using liquid phase extraction with back extraction combined with high performance liquid chromatography

Liquid phase extraction with back extraction (LPE-BE) combined with high performance liquid chromatography-diode array detection (HPLC-DAD) was applied for the extraction and determination of erythromycin A, B and C in fermentation broths. According to this procedure, the fermentation broth with the adjustment pH at a fixed value of 10 was first mixed with organic solvent (V_broth/V_org = 1.0). After shaking, the mixture was separated into two phases by microfuging at 13,000 rpm for 15 min. Then back extraction was performed into the acidic aqueous phase with pH 5.0 (V_org/V_aq = 1.0). After centrifugation at 3000, the two phases were separated and 50 μL of the acidic aqueous phase was injected into the HPLC. The effects of different variables such as the nature of extraction solvent and the pH of samples and buffer were investigated. At the most appropriate conditions, dynamic linear ranges of 0.5–8, 0.1–0.9 and 0.1–0.9 mg mL⁻¹ and limits of detection of 0.03, 0.003 and 0.002 mg mL⁻¹ were obtained for erythromycin A, B and C, respectively. Relative standard deviations (RSDs) of the proposed method were less than 9.5%. The mean recoveries were 99.5%. The proposed method is simple and sensitive with highly clean-up effect and it can be used for monitoring the progress of erythromycin fermentation.     

A rapid HPLC determination of narasin in fermentation broth

Summary A rapid HPLC assay with post-column derivatization has been developed for the determination of narasin in fermentation broth. The reaction of narasin with substituted benzaldehydes was investigated under first order conditions and the rate constants were determined for a variety of substituted benzaldehydes. Vanillin reacted most rapidly to produce a red color. The reaction conditions were optimized to acheive a maximum response with a minimum analysis time.     

High-performance liquid chromatographic determination of (−)-cathinone in plasma

Summary High-performance liquid chromatography (HPLC) for the determination of (–)-cathinone in rabbit and human plasma has been studied. The problem of dimerization during extraction from plasma was satisfactorily resolved. Detection was by UV at 257 nm. Concentration levels as low as 24 ng ml^–1 were satisfactorily determined. This level of sensitivity should be adequate for the detection of (–)-cathinone in the blood of khat users and also for the quantitative determination of (–)-cathinone in blood for pharmacokinetic purposes. The applicability of the assay procedure to pharmacokinetic studies is demonstrated.     

Liquid chromatographic determination of fluvastatin and its enantiomers in blood plasma by automated solid-phase extraction

Summary Liquid chromatographic methods for determination of fluvastatin, as racemate and asseparated enantiomers, are described. Fluvastatin is an anticholesterolemic agent that inhibits the rate limiting enzyme in cholesterol biosynthesis. The sample clean-up was fully automated, using solid-phase extraction. Plasma samples were injected onto disposable C2 cartridges, connected on-line with the analytical column. After washing, the analytes were eluted and transferred to a C8-analytical column for racemate determination or to a Chiralcel OD-R column for enantiomer separation. Analytes were monitored by fluorescence detection after post-column exposure of the eluate to UV light, which enhanced the sensitivity 20-fold for fluvastatin and 10-fold for the enantiomers. Absolute recoveries were >90% both for the recemate and enantiomers. Limits of quantitation were 0.5 nM for the racemente and 1 nM for enantiomers, using 200 μL plasma sample. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

High-Performance liquid chromatographic determination of dibromomannitol in plasma

Summary Myelobromol 1,6-dibromo-1,6-dideoxy-D-mannitol (dibromomannitol, DBM) is a bifunctional alkylating agent that has been in clinical use since 1963. It is currently included at high dose in preconditioning regimen for bone marrow transplantation (BMT) and is a main-stay of treatment for polycythaemia vera. A high-performance liquid chromatographic method was developed for the determination of DBM in the plasma. The basis of the assay is a derivatization with sodium-diethyldithiocarbamate at 42°C in the presence of 1-bromo-1-deoxy-3,6-anhydro-galactitol as internal standard (IS). The analysis was carried out on a 250×4mm Hypersil 5 CPS column equipped with a 20×4 mm Hypersil 10 CPS precolumn. The eluent consisted of heptane:isopropyl-alcohol: glacial acetic acid=600:76:80 w/w. The flow rate was 1.2 mL min⁻¹. UV detection was performed at 254 nm. The calibration graph was linear in the concentration range of 2.5–260 μM of DBM in plasma. The limit of detection was 1.0 μM. The precision and accuracy of the method was between the good laboratory practice (GLP) required limits. Presented at: Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 3–5, 1997     

Determination of tylosin and related macrolides in fermentation broth by gradient elution ion-pair liquid chromatography

Summary A gradient elution ion-pair HPLC assay was developed for the analysis of tylosin and related macrolides in fermentation broth. The effect of the ion-pair agent concentration and identity was studied to optimize the reproducibility of the separation. The assay compares favorably with the biological assay and is sufficiently durable for the analysis of many samples per column.     

Liquid chromatographic analysis with electrochemical detection for metoclopramide in human plasma

Summary A simple, reproducible liquid-chromatographic method for determination of metoclopramide anti-emetic and anti-spasmodic agent is described. Metoclopramide is extracted from alkalinized plasma into diethylether, then separated with a sperisorb CN column and measured through amperometric detection at +1,4 volt vs Ag/ AgCl. The average extraction recovery was 92%. Standard curves were linear and reproducible from 0.05 to 1.5 mg/ L. Within-day CV was better than 6%.     

Liquid chromatographic analysis of sulfornamides in foods

Summary A procedure for the simultaneous determination of several sulfonamides in different foods, such as honey, milk and eggs is proposed. The analysis is carried out using reversed phase liquid chromatography with spectrophotometric detection. Optimization of the mobile phase led to good separation and a short analysis time when an initial isocratic step with a 397 acetonitrile: water mixture was used for 5 minutes, followed by a linear gradient up to a 4060 mixture over 15 min. The proposed method is suitable for routine quality control analysis to ensure the absence of sulfonamides in foods. Recovery studies yielded good results for all food samples because there were no interferences from the matrices.     

High-performance liquid chromatographic determination of busulfan in plasma

Summary Busulfan (Myleran; 1,4-bis-(methanesulfonyloxy) butane; BU) is a bifunctional alkylating agent used in clinical practice since 1959. It is currently included at high doses in conditioning regimens for bone marrow transplantation, usually in combination with cyclo-phosphamide. A high-performance liquid chromatographic method has been developed for the determination of BU in plasma. The basis of the assay is a derivatization with sodium diethyldithiocarbamate at 32°C in the presence of 1-bromo-1-deoxy-3,6-anhydrogalactitol as internal standard. Analysis is performed on a cyano column with heptane-isopropanol-glacial acetic acid as mobile phase and UV detection at 280 nm. The calibration graph was linear in the concentration range 0.18–46.40 μM BU in plasma. The limit of detection was 0.1 μM. The precision and accuracy were between the limits required by good laboratory practice. Presented at Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, september 1–3, 1999     

Liquid chromatographic behaviour of pyrimidine bases on various bonded phases

Summary The effect of the surface chemistry of bonded-phase column material containing alkyl, phenyl, cyano and amino groups on the adsorption of pyrimidine molecules has been studied by HPLC. The dependence of the retention on the chemical structure of the adsorbed molecules was determined. The influence of the substituents at the pyrimidine ring and their position on the retention character is shown for various bonded phases.     

Determination of monensins A and B in the fermentation broth ofStreptomyces cinnamonensis by high performance liquid chromatography

Summary High performance liquid chromatography on an octadecyl silica column has been used to determine both the monensin A: monensin B ratio and by the method of standard addition, the concentration of both in the fermentation broth ofStreptomyces cinnamonensis. Refractive index detection was preferred to ultraviolet owing to the presence of UV-absorbing components which could not be completely separated from the substances of interest. A linear relationship was obtained from the calibration data. The coefficients of variation for the estimation both of the ratio and the concentrations of the compounds were better then 5%. The estimated limit of detection for both substances was about 1 g/ml. The results obtained from the determination of the ratios of monensins were compared with those obtained by chemical ionization mass spectrometry. Chromatographic separation of monensins on the silica gel column is also described.     

High-performance liquid chromatographic determination of urocanic acid isomers in cosmetic products

Summary A method has been developed for the determination of trans and cis urocanic acid in oil-in-water cosmetic emulsions. It involves an extraction of the sample in 1:3 methanol-aqueous NaOH (10⁻³ M), by ultrasonication, which leads to quantitative recoveries, and a reversed-phase HPLC isocratic elution for the analysis of the extract. Chromatography is performed on a C18 column using 0.1 M sodium perchlorate (pH 3.0)-acetonitrile 98:2 (v/v), as the mobile phase, and UV detection at 263 nm. The separation of the isomers is good. Linearity and precision of the method have been assessed.     

Automated high-performance liquid chromatographic determination of amphetamine in biological fluids using column-switching and on-column derivatization

Summary A rapid and simple liquid-chromatographic method has been developed for on-line quantification of amphetamine in biological fluids. Untreated samples (20 μL) are injected directly into the chromatographic system and purified on a 20 mm×2.1 mm i.d. pre-column packed with 30 μm Hypersil C₁₈ stationary phase. After clean-up the analyte is transferred to the analytical column (125 mm×4 mm i.d., 5 μm LiChrospher 100 RP₁₈) for derivatization and separation using a mixture of acetonitrile and the derivatization reagent (o-phthaldialdehyde andN-acetyl-L-cysteine) as the mobile phase. The experimental conditions for on-line derivatization and resolution of the amphetamine have been optimized, and the results have been compared with those obtained by derivatizing the analyte in pre-column mode. The method described has been applied to the determination of amphetamine in plasma and urine. Good linearity and reproducibility were obtained in the 0.1–10.0 μg mL⁻¹ concentration range, and limits of detection were 25 ng mL⁻¹ and 10 ng mL⁻¹ with UV and fluorescence detection, respectively. The procedure described is very simple and rapid, because no off-line manipulation of the sample is required; the total analysis time is approximately 8 min.     

High-performance liquid chromatographic determination of glaucine inGlaucium flavum crantz

Summary A method has been developed for the extraction and rapid analysis of D-glaucine inGlaucium flavum Crantz. Simple extraction of the drug with diethyl ether was followed by high-performance liquid chromatography on a μBondapak C₁₈ column using a mixture of acetonitrile, methanol and phosphate buffer as the mobile phase. Data on selectivity, sensitivity and precision demonstrate the reliability of this method.     

Liquid chromatographic determination of nitroaromatics in water following solid phase extraction,pre-column reduction and derivatization

Summary A selective and sensitive method for determination of nitroaromatics is described. The analytes were reduced to corresponding primary amines with iron powder and then derivatized with fluram in citrate buffer to form pyrrolinones. The highly fluorescent pyrrolinones were isolated and preconcentrated by octadecylsilane solid phase extraction cartridge followed by reversed-phase, high-performance liquid chromatographic analysis. Detection was at 395–495 nm. Various aspects such as the reduction process, derivatization, solid phase extraction and chromatographic separation were optimized. Analysis time was relatively short due to a special design for successive reduction and preconcentration. Limits of detection for 3-nitrophenol, nitrobenzene, 4 and 2-nitrotoluene were less than 60, 12, 60 and 280 ng L^–1 respectively.     

High-performance liquid chromatographic determination of meropenem in rat plasma using column-switching

Summary The purpose of this study was to develop a columnswitching HPLC method for the determination of meropenem in plasma. This method showed excellent precision and accuracy with good sensitivity and speed. The total analysis time per sample was less than 20 min and the mean coefficients of variation for intra- and inter-assay were less than 4.0%. The method has been successfully applied to plasma samples from rats receiving an intraperitoneal injection of meropenem.     

Liquid chromatographic determination of aliphatic amines in water using solid support assisted derivatization with 9-fluorenylmethyl chloroformate

Summary A simple and sensitive method has been developed for the liquid chromatographic determination of short-chain aliphatic amines in water. Analytes are retained in solid-phase extraction (SPE) cartridges, and then derivatized by drawing an aliquot of the fluorogeneic reagent 9-fluorenylmethyl chloroformate (FMOC) through the cartridges. After a certain reaction time the derivatives formed are desorbed with acetonitrile. The collected extracts are then chromatographed on a LiChrospher 100 RP₁₈ 125 mm×4 mm i.d., 5 μm, column using an acetonitrile-water gradient. The influence of experimental conditions (SPE material, volume of sample, concentration of FMOC, time of reaction and pH) has been investigated. Optimal results have been obtained with C₁₈ SPE cartridges using a sample volume of 5.0 mL. For derivatization, 0.25 mL aliquots of 25 mM FMOC have been used, the reaction time being only 2 min. The method has been applied to the quantification of several aliphatic amines: methylamine, ethylamine, dimethylamine,n-butylamine,n-pentylamine andn-hexylamine. Under the proposed conditions the percentages of analytes retained plus derivatized were of about 54–107% compared to those obtained with direct solution derivatization. The method provided good reproducibility, linearity and accuracy within the 0.050–1.0 mg L⁻¹ concentration range. The limits of detection were in the 0.25–5.0 μg L⁻¹ range. The utility of the described approach has been tested by analysing tap water, river water and industrial waste water.     

Liquid chromatographic separation of fluoroquinolone antibacterials used as veterinary drugs

Summary A liquid chromatographic method has been developed to separate a series of quinolones which are widely used as veterinary drugs: danofloxacin, difloxacin, enrofloxacin (and its metabolite ciprofloxacin), marbofloxacin, norfloxacin and sarafloxacin. Separation was performed by using an end-capped high-purity silica-based C₈ column and a mobile phase consisting of acetonitrile-aqueous oxalic acid buffer solution. In order to establish the optimal conditions for an isocratic separation, a three-factor Doehlert experimental design was applied. Gradient elution was applied which reduced analysis time. Figures of merit of the method proposed, with fluorimetric detection, were evaluated.     

Isocratic separation of erythromycin, related substances and degradation products by liquid chromatography on XTerra RP18

Summary A simple, sensitive, selective and robust isocratic LC method is described for the analysis of erythromycin on XTerra RP₁₈. The main component, erythromycin A, is separated from all known related substances and degradation products. Several unknown impurities are also separated. Acetonitrile-0.2 MK₂HPO₄pH7.0-water, (35∶5∶60, v/v) was used as a mobile phase at 1.0 mL min⁻¹. UV detection was at 215 nm. The robustness of the method was evaluated by a full-factorial experimental design.     

Liquid chromatographic determination of aniline and derivatives in environmental waters at nanogram per litre levels using fluorescamine pre-column derivatization

Summary Fluorescamine (fluram) has been used as a fluorogenic compound for pre-column derivatization of aniline and some derivatives. Anilines were derivatized with fluram in citrate buffer media (pH 5.5) to form pyrrolinones. The highly fluorescence pyrrolinones were isolated and pre-concentrated by solid phase extraction. A reversed phase, Spherisorb RP-8 column and tetrahydrofuran: water:formic acid (42:56:2) mobile phase was used for separation. Detection method was by a sensitive fluorimetric method and quantitation was at 395 and 495 nm. The various parameters such as reaction conditions between anilines and fluram, solid phase extraction and chromatographic separation were optimized. Calibrations were linear over the range considered with excellent correlation coefficients (r>0.999). Relative standard deviations are less than 2.5 % and detection limits for aniline,p-toluidine, 4-chloroaniline and 4-bromoaniline were 6, 30, 6 and 8 ng L⁻¹, respectively. This method has been used successfully for the determination of anilines in environmental waters.