A chip-based immunoaffinity capillary electrophoresis assay for assessing hormones in human biological fluids

A chip-based capillary electrophoresis system has been designed for assessing the concentrations of four hormones in whole human blood, saliva, and urine. The desired analytes were isolated by immunoextraction using a panel of four analyte-specific antibodies immobilized onto a glass fiber insert within the injection port of the chip. Following extraction, the captured analytes were labeled prior to electro-elution into the chip separation channel, where they were resolved into four individual peaks in circa 2 min. Quantification of each peak was achieved by on-line LIF detection and integration of the area under each peak. Comparison to commercial high-sensitivity immunoassays demonstrated that the chip-based assay provided fast, accurate, and precise measurements for the analytes under investigation. As the availability of commercially available antibodies rapidly expands, the application of this system will greatly increase. Chip-based CE separations of multiple analytes from a single sample also provide a significant advantage in the analysis of small samples.     

A rapid and specific high-performance liquid chromatographic assay for theophylline in biological fluids

A rapid, specific high-performance liquid chromatographic analysis of theophylline in plasma, serum, and saliva is described. Proteins present in the biological samples are precipitated with 6% perchloric acid and the clear supernatant is chromatographed on a reversed-phase column. Only 100 microL of serum is required and concentrations as low as 0.07 micrograms/mL can be measured accurately. Other xanthines do not interfere in the assay. Within- and between-day variation is less than or equal to 2.2%. The method shows less bias and greater precision than the TDx (Abbott Diagnostics) procedure commonly used in clinical laboratories.     

A simple spectrophotometric procedure for determination of valaciclovir in dosage forms and biological fluids

A simple and selective spectrophotometric procedure for the determination of Valaciclovir (VALA) is proposed and applied to determine the substance in tablets and biological fluids. This procedure is proposed for the determination of VALA in Britton-Robinson (BR) buffer at pH 6.0, which allows quantification over the 4 × 10^?6 to 6 × 10^?5 M range for spectrophotometric measurements. The repeatability and reproducibilty of the procedure for serum and breast milk samples are estimated. Precision and accuracy are also checked. The standard additions method is used for biological fluids. The proposed produce was verified for the determination of VALA in real human serum, breast milk samples; the selectivity over the metabolites was found to be quite satisfactory.     

A new quantitative thermospray LC-MS method for nicotine and its metabolites in biological fluids

A rapid thermospray liquid chromatography-mass spectrometry (TSP LC-MS) method is described for the simultaneous determination of nicotine and 17 of its metabolites. Chemical ionization of nicotine and its metabolites separated by reversed-phase HPLC is achieved by postcolumn addition of ammonium acetate buffer with the filament of the ion source turned off. Quantification is accomplished by selectively monitoring the unique protonated molecular ion of each metabolite. Trideuterated cotinine serves as an internal standard. Linear responses for cotinine, demethylcotinine, and trans-3'-hydroxycotinine were observed over a concentration range of 20-8000 ng/mL, and 80-8000 ng/ml for nicotine and nicotine-1'-N-oxide. Of the 17 metabolites examined, only nicotine, cotinine, demethylcotinine, and trans-3'-hydroxycotinine were detected in smokers' urine.     

Methodology for isolating drug metabolites from biological fluids by HPLC

This article presents a detailed account of the isolation of drug metabolites from bile by reverse-phase HPLC. The systematic approach is illustrated for the metabolism of butoprozine, a new antianginal drug, in the dog. Separation utilizes isocratic and gradient elution. Detection of the metabolites in. the presence of endogenous bile components is accomplished by very selective radioactivity measurements, by less selective UV (single wavelength) detection, and by much more universal detection modes such as on-line UV spectroscopy and off-line mass- and nuclear magnetic resonance spectroscopy. The importance of the latter three techniques in purity analysis of isolated metabolites is illustrated.     

HPLC measurement of penticainide and desalkylpenticainide in biological fluids

A simple HPLC method has been developed to measure the antiarrhythmic agent penticainide and its N-desalkyl metabolite in biological fluids. Solvent extraction of a small (200 microL) sample volume with direct analysis of the extract is used to measure the plasma and urinary concentrations of these compounds attained during chronic therapy, although a larger sample volume (1.0 mL) and prior concentration of the extract are required for single oral dose work. In each case chromatographic analysis is performed using a microparticulate (5 microns) silica column and methanolic ammonium perchlorate (10 mM, pH 6.7) as eluent with UV detection (260 nm). No endogenous sources of interference have been encountered and potential interference from other drugs is minimal.     

Estimation of diuretic drugs in biological fluids by HPLC

Summary This critical review of different methods proposed for the determination and screening of diuretics is directed mainly, because of its potential application, towards highperformance liquid chromatography.     

HPLC assay for determination of amphotericin B in biological samples

A fast and selective HPLC method for assaying amphotericin B in biological samples was developed and validated. The chromatographic separation was achieved in less than 12 min on a reverse-phase C(18) column using an acetonitrile-acetic acid-water (52:4.3:43.7, v/v/v) mixture as mobile phase. The flow rate was 1 mL/min and the effluent was monitored at 406 nm. A linear response over the concentration range 0.1-10.0 microg/mL was obtained. Intra-day and inter-day RSDs were below 5% for all the sample types. This new HPLC method was applied to assay amphotericin B in plasma and several tissue samples such as kidney, liver, spleen and bone marrow. Application of this method to pharmacokinetic studies in mice and dog is provided.     

Solid phase extraction for an improved assay of physostigmine in biological fluids

A simple, selective and very sensitive assay is described for the quantification of physostigmine in blood, plasma and urine. The most appropriate solid phase column was selected after a systematic investigation of nine types of phase. The conditions for solid phase extraction were optimized using [3H]physostigmine so that the overall recoveries were greater than 90%. Physostigmine was retained on alkaline treated cyanopropyl columns and eluted into the minimum volume of methanol, obviating the need for an evaporation step. Extracted samples were quantified by HPLC with a three electrode coulometric detection system. The limit of detection was 50 pg/mL for a 0.5 mL plasma sample. The precision (CV) for 0.5 mL plasma samples containing 50 pg was 8.1%. Application of the method to plasma, blood and urine samples is presented.     

Development of enantioselective gas chromatographic quantitation assay for dl-threo-methylphenidate in biological fluids

An enantioselective gas chromatographic quantitation assay was developed for the enantiomers of dl-threo-methylphenidate in plasma and urine. dl-threo-Methylphenidate and the internal standard were acylated with N-heptafluorobutyryl-1-prolylchloride under Schotten-Baumann conditions prior to gas chromatographic separation on achiral mixed stationary phases. The derivatives were detected by means of a nitrogen-phosphorus detector. Linear and reproducible calibration curves were obtained over the concentration ranges 0.43-43.25 and 2.16-216.25 ng/ml enantiomer in plasma or urine, respectively. This enantioselective gas chromatographic quantitation assay was applied in a single oral dose disposition study of dl-threo-methylphenidate in a healthy adult volunteer. Stereoselective differences were observed in the plasma concentration-time profiles and cumulative urinary excretion profiles following oral doses of 20 and 40 mg of dl-threo-methylphenidate hydrochloride. Only d-threo-methylphenidate was detectable in plasma after 4 h.     

Multiple inverse isotope dilution assay for the stereospecific quantitative determination of R(-) and S(+)-oxaprotiline in biological fluids

An isotope dilution assay for the determination of both oxaprotiline enantiomers in biological samples after administration of the racemic mixture has been developed. The enantiomers were reacted with synthetically prepared, optically pure N-trifluoroacetyl-S(-)-prolyl chloride, followed by high-performance liquid chromatographic separation of the diastereoisomers formed. Quantitation was performed by on-line UV detection at 260 nm and off-line radiometry by liquid scintillation counting. Endogenous compounds and metabolites do not interfere in the assay. Analysis of water and the blood and urine of rats spiked with [14C]oxaprotiline X HCl showed recoveries for S(+)-oxaprotiline X HCl (mean +/- coefficient of variation, n = 4-6) of 98.0 +/- 1.0% (water), 100.5 +/- 0.6% (blood) and 101.5 +/- 2.0% (urine), and for R(-)-oxaprotiline X HCl of 101.3 +/- 2.0% (water), 102.2 +/- 2.1% (blood) and 103.2 +/- 0.2% (urine). A pilot study to determine blood levels of the two enantiomers in two rats dosed with racemic [14C]oxaprotiline X HCl (10 mg/kg i.v.) was carried out to test the method. The results indicated stereoselective disposition of oxaprotiline enantiomers in the rat. The ratio of the areas under the blood concentration curves for R(-)-to S(+)-oxaprotiline X HCl was 1.14.     

Liquid-solid extraction and HPLC determination of amiodarone metabolites in biological fluids

Summary An improved liquid-solid extraction procedure for the purification and determination of amiodarone and desethylamiodarone in biological fluids is proposed. The sample is passed through a silica-C₁₈ column, and after washing, the analytes are eluted with methanol. The determination is then accomplished by RP HPLC using an octadecyl silica column and a mobile phase of methanol containing 0.0015% of ammonium hydroxide. The effect of ammonia concentration on the capacity factors of the analytes has been used for estimating the acid dissociation constants of the investigated secondary and tertiary amines in methanol.     

Quantitative assay of maprotiline in biological fluids by gas-liquid chromatography.

A specific and sensitive gas-liquid chromatographic method has been developed for the assay of maprotiline in biological fluids. Maprotiline is isolated from the biological sample by base-specific extraction followed by conversion into the heptafluorobutyramide. The derivative is determined quantitatively by gas-liquid chromatography with an electron capture detector, nortriptyline being used as the internal standard. Amounts below 10 ng per biological sample can be measured.