High-performance liquid chromatographic analysis of dinitrobenzene isomers

Summary An efficient, reproducible and rapid high-performance liquid chromatographic method, in normal phase mode, for the analysis of the three dinitrobenzene isomers is described. The method affords good linearity for each isomer in the range 10–160 g ml^–1. The total analysis time is only 10 minutes, and the method shows an accuracy of ±1.25% with a coefficient of variation from 0.30% to 2.85% for different levels of the dinitrobenzene isomers.     

High-performance liquid chromatographic analysis of azlocillin in serum

Summary A rapid and sensitive high performance liquid chromatographic method is described for the determination of azlocillin in serum. This method involves a short manual protein precipitation of the sample followed by an injection into a PR 18 column for separation and quantitation. The mobile phase was a 22% (V/V) solution acetonitrile in phosphate buffer pH 4.8 at a flow rate of 2,5 ml/min. The spectrophotometer detector was set at 220 nm with a sensitivity of 0.08 AUFS.     

High-performance liquid chromatographic assay of bacterial collagenase

Summary The activity of bacterial collagenase Clostridiopeptidase A was estimated using a labelled synthetic peptide, 4-phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg, as substrate. The N-protected dipeptide obtained after enzymatic hydrolysis of Leu-Gly peptide bond was quantified by reversed-phase, high-performance liquid chromatography using 4-phenylazobenzyloxycarbonyl-L-Pro-L-Phe as internal standard. The time dependence of the appearance of the hydrolysis product and the dependence of rates of hydrolysis on collagenase concentration were linear. Kinetic parameters for collagenase were determined to test the suitability of the described procedure.     

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.     

High-performance liquid chromatographic measurement of 8-methoxypsoralen in plasma

Summary A simple high-performance liquid chromatographic method for the measurement of 8-Methoxypsoralen (8-MOP) in human plasma following a single 40mg dose has been described. After addition of phosphate-NaOH buffer, pH 12, and internal standard (trimethylpsoralen), the sample is vortex-mixed with diisopropylether. The resulting extract is analysed on a reverse phase column using phosphoric acid (0.05% v/v): acetonitrile (1:1) as mobile phase, and U.V. detection at 220nm. No interference from endogenous sources has been observed. The limit of sensitivity of the assay is 5ng/ml plasma. The measuring range is between 10–700ng 8-MOP/ml plasma, to be expected from oral doses of 0.6mg 8-MOP/kg body weight, and corresponds to the therapeutic plasma concentration. The relative standard deviation at 50ng/ml level of 8-MOP is 3.6%.     

High-performance liquid chromatographic separation of hematoporphyrin and its nickel,copper and zinc complexes on octadecyl-bonded stationary phase

Summary The feasibility of reversed-phase high-performance liquid chromatography for the separation of metal complexes of hematoporphyrin IX (Hp) is described. The retention order, Zn-complex<Hp (free acid)< Ni-complex<Cu-complex, is regular on an octadecylbonded stationary phase with different compositions of an aqueous methanol mobile phase. These four compounds can be successfully separated within about 8 min on a LiChrosorb RP-18 column (250×4-mm i.d.) with a 85:15 (vol/vol) mixture of methanol and phosphate buffer (pH 3) at a flow rate of 1 ml/min.     

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     

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.     

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.     

High performance liquid chromatographic separation of cholesterol oxidation products

Summary A fast, sensitive, high performance liquid chromatographic method for the simultaneous determination of cholesterol hydroperoxides and other major oxysterols, using two different detection systems (ultraviolet at 210 nm and light scattering), is here described. The hydroperoxy derivatives were obtained by cholesterol photoxidation, isolated by thin layer chromatography and joined with a standard mixture of 10 oxysterols (epoxy, hydroxy and keto derivatives). Aliquots were directly injected onto a 5-μm particle size, 25×0.46 cm i.d. Spherisorb S5 CN normal phase column, usingn-hexane/anhydrous ethanol (97:3, v/v) as mobile phase and a flow rate of 0.8 mL min⁻¹. This method allowed, in a single isocratic analysis, the separation and quantification of the primary and secondary cholesterol oxidation products in 30 minutes. The light scattering detector was particularly useful for the determination of nonderivatized 5,6-epoxides and cholestane-3β,5α,6β-triol. The sensitivity of both detectors was very similar for most of the oxysterols, except for the 5,6-epoxides and the 7-ketocholesterol. The method suitability for the determination of cholesterol oxidation products in food matrices was successfully tested on a saponified lipid extract from egg yolk powder.     

High-performance liquid chromatographic resolution of enantiomers on chiral amine-bonded silica gel

Summary A chiral stationary phase with an immobilized, optically active diamine was prepared for the separation of enantiomers. The synthesis of the phase was carried out by bonding (–)trans-1,2-cyclohexanediamine to microparticulate silica gel through the coupling agent 3-glycidoxypropylsilane. The resolution of the racemic compounds catechin, 2,2-dihydroxy-1,1binaphthyl and trans-1,2-cyclohexandiol, is reported.     

High-performance liquid chromatographic determination of furfural and hydroxymethylfurfural in apple juices and concentrates

Summary A rapid and sensitive method for determining 2-furaldehyde (FUR) and 5-hydroxymethyl-2-furaldehyde (HMF) in apple juices and juice concentrates has been developed. The method for FUR and HMF involves the solid-liquid extraction of the juice by using a C-18 cartridge prior to reversed-phase separation with detection at 280 nm. The mobile phase was acetonitrile-water (8/92, v/v) at a flow rate of 1.0 ml/min. Recoveries from apple juices and juice concentrates spiked at different levels ranged from 94.1 to 104.0 (FUR) and 94.5 to 100.5 (HMF). The quantification limit for both, FUR and HMF, was 5 ppb.     

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.     

High-performance liquid chromatographic analysis of isoxazolylpenicillins in plasma and urine samples

Summary A simple and fast liquid chromatographic method is described, applicable to the routine analysis of isoxazolylpenicillins (cloxacillin, dicloxacillin, flucloxacillin) in biological fluids (plasma, urine). The method is based on a simple dilution step employed to destroy the protein binding, which is over 95%, and allows the detection of concentrations down to 10μg/ml. In order to analyze concentrations of less than 10μg/ml, a liquid-liquid extraction with dichloromethane must be executed prior to the reversed-phase analysis with absorbance detection at 206nm. The minimum detectable amounts of the isoxazolylpenicillins with this procedure are between 2.5 and 5.1 ng in 100μl plasma samples. The stability of the penicillin samples in aqueous solutions (stock solutions, eluents) was investigated and no significant degradation was observed during the storage and analysis of the samples. Furthermore, the degree of protein binding was established by using a suitable ultrafiltration technique, and the usefulness of the developed procedures in pharmacokinetic studies was demonstrated.     

High-performance liquid chromatographic separation of polyolefin antioxidants and light-stabilisers

Summary A scheme was devised for the identification of 22 common antioxidants and light-stabilisers in polyolefins. The separation of these stabilisers was performed by isocratic reversed phase high-performance liquid chromatography on a RP-18 column. Three different separation conditions have been used: the mobile phase composition was 100% acetonitrile (MeCN), 90/10 meCN/H₂O and 80/20 MeCN/H₂O. The UV₂₅₄/UV₂₈₀ ratio and the elution time of each stabiliser were determined for these three mobile phase compositions. The values of UV₂₅₄/UV₂₈₀ ratios may be used together with the retention time values for the identification of unknown stabilisers in polyolefin samples.     

High-performance liquid chromatographic separation of BisGMA oligomers and isomers in dental restorative materials

Summary The composition of the dental monomer BisGMA was analyzed using various techniques of high-performance liquid chromatography (HPLC): isocratic and gradient-elution, normal-phase and reversed-phase-HPLC. These techniques emphasized that BisGMA is not a single compound corresponding to the well known structure of 2,2-Bis[4-(2-hydroxy-3-methacryloyloxypropoxy)phenyl]propane and allowed the separation of BisGMA oligomers and isomers in dental restorative materials. The analysis of the dental resins emphasized the presence of three isomers for each BisGMA oligomer.     

Development and validation of an improved liquid chromatographic method for the analysis of oxytetracycline

Summary An improved LC method is described for the separation of oxytetracycline and its impurities. The separation is much better than that obtained with official pharmacopoeia methods. The method uses XTerra RP-18, 5 μm (25cm×4.6 mm I.D.), a silica-based stationary phase with methyl end-capping, claimed to reduce silanol activity. The column temperature is set at 30°C and a UV detection is performed at 280 nm. Mobile phase containing acetonitrile −0.25 M tetrabutylammonium hydrogen sulfate pH 7.5−0.25 M ethylenediaminetetraacetic acid pH 7.5-water (115:360:160:365,v/v/v/v) is used at a flow rate of 1.0 mL.min⁻¹, to separate the impurities present in oxytetracycline base. A central composite experimental design is used to optimize the separation. A second isocratic method with higher content of acetonitrile is needed to separate the more retained impurities present only in oxytetracycline hydrochloride. The method is robust and shows good selectivity, repeatability, linearity and sensitivity.     

High-performance liquid chromatography separation of chlordecone and its metabolites

Summary High-performance liquid chromatography (HPLC) was used to separate chlordecone (Kepone), and two of its metabolites, hydrochlordecone and dihydrochlordecone. Elution of the three peaks occured after the solvent concentration reached 100% methanol and was maintained at 100% for approximately five minutes. The method was linear for chlordecone in the concentration range of 10 to 100 g.     

A micellar liquid chromatographic method for quality control of pharmaceutical preparations containing tricyclic antidepressants

Summary Micellar liquid chromatography methods for quality control of pharmaceutical preparations (capsules, pills, tablets, injections) containing the tricyclic antidepressants amineptine, amitriptiline, clomipramine, doxepin, imipramine, melitracen and nortriptyline alone or together with other CNS drugs like diazepam, medazepam and perphenazine are described. The methods using micellar solutions of cetyltrimethylammonium bromide as mobile phases and UV detection are rapid and reproducible. Due to the versatility of interactions in micellar liquid chromatography, it is possible determine highly hydrophobic compounds such as TCAs in a short time using mobile phases containing low organic solvent concentrations and usual flow rates, in contrast with the RP-HPLC methods proposed for these compounds. Samples preparation only requires solution and adequate dilution with the mobile phase before injection into the chromatographic system.