Determination of methacrylonitrile in serum using gas chromatography and flame ionization detection.

A gas chromatographic method is described for the quantitation of methacrylonitrile in serum. Methacrylonitrile was extracted from rat serum with diethyl ether and then quantified using a gas chromatograph equipped with a flame ionization detector and a 60-m megabore column coated with polyethylene glycol polymer. The recoveries obtained following a one-step extraction with diethyl ether varied from 60% at 3.2 micrograms/ml to 70% at 80 micrograms/ml. The coefficient of variation for the analysis ranged from 2.5% at 400 micrograms/ml to 15.0% at 3.2 micrograms/ml.     

High-performance liquid chromatographic analysis of isoxicam in human plasma and urine

A sensitive, selective, and rapid high-performance liquid chromatographic procedure was developed for the determination of isoxicam in human plasma and urine. Acidified plasma or urine were extracted with toluene. Portions of the organic extract were evaporated to dryness, the residue dissolved in tetrahydrofuran (plasma) or acetonitrile (urine) and chromatographed on a mu Bondapak C18 column preceded by a 4-5 cm X 2 mm I.D. column packed with Corasil C18. Quantitation was obtained by UV spectrometry at 320 nm. Linearity in plasma ranged from 0.2 to 10 micrograms/ml. Recoveries from plasma samples seeded with 1.8, 4 and 8 micrograms/ml isoxicam were 1.86 +/- 0.077, 4.10 +/- 0.107 and 8.43 +/- 0.154 micrograms/ml with relative standard deviations of 3.3%, 2.5% and 5.4%, respectively. The linearity in urine ranged from 0.125 to 2 micrograms/ml. The precision of the method was 3.3-9.0% relative standard deviation over the linear range.     

Determination of cholesterol and cortisone absorption in polyurethane. I. Methodology using size-exclusion chromatography and dual detection.

A size-exclusion chromatographic method is described for measuring the absorption of the steroid-based lipids cholesterol and cortisone into Pellethane 2363, a polyurethane used in biomedical implants. The method uses refractometry and ultraviolet diode-array detection, with tetrahydrofuran as the mobile phase. Using an injection volume of 150 microliters, the lower limit of accurate measurement for cholesterol (refractive index detection) was 6 micrograms/ml with a lower limit of detection, based on a 2:1 signal-to-noise ratio, of 0.15 micrograms (1 microgram/ml). For cortisone (ultraviolet detection), the lower accurate limit was 0.6 micrograms/ml with a lower limit of 0.015 micrograms (0.1 micrograms/ml). The results show that after 44 h, 2037 micrograms/g cholesterol and 3131 micrograms/g cortisone were absorbed by the polyurethane. The method eliminates extensive sample manipulation and is sensitive to low levels of lipid in the presence of a high-molecular-mass synthetic polymer.     

High-performance liquid chromatographic analysis of amphotericin B in plasma, blood, urine and tissues for pharmacokinetic and tissue distribution studies.

A sensitive and reproducible high-performance liquid chromatographic method was developed to assay ampherotericin B in plasma, blood, urine and various tissue samples. Amphotericin B was isolated from each sample matrix by solid-phase extraction (Bond-Elut). The eluate from Bond-Elut containing amphotericin B was injected onto a reversed-phase C18 column (Waters, mu Bondpak, 10 microns, 300 mm x 3.9 mm I.D.) with a mobile phase of 45% acetonitrile in 2.5 mM Na2EDTA at 1 ml/min. Detection of amphotericin B was by ultraviolet absorption at 382 nm. Blood and tissues were homogenized and extracted with methanol prior to Bond-Elut extraction. The extraction efficiencies of amphotericin B from plasma, blood and tissues were approximately 90, 70 and 75%, respectively. The sensitivity of the assay was less than or equal to 5 ng/ml for plasma, less than or equal to 25 ng/ml for blood, 2.5 ng/ml for urine and 50 ng/g for tissues. The linearity of the assay method was up to 2.5 micrograms/ml for plasma, 5 micrograms/ml for blood, 500 ng/ml for urine and 500 micrograms/g for tissues. The assay was reproducible with an intra-day coefficient of variation (C.V., n = 3) of less than 5% in general for plasma, blood and tissues. The inter-day C.V. of the assay was less than 5% for plasma (n = 5), less than 10% for blood (n = 4) and less than 5% for tissues (n = 3). The overall variability in the urine assay was generally less than 10%. This method has demonstrated significant improvement in the sensitivity and reproducibility in assaying amphotericin B in plasma and especially in blood, urine and tissues. We have employed this assay to compare the pharmacokinetic and tissue distribution profiles of amphotericin B in rats and dogs following administration of Fungizone and ABCD (amphotericin B-cholesteryl sulfate colloidal dispersion), a lipid-based dosage form. In addition, the assay method for plasma and urine samples can also be applied to pharmacokinetics studies of amphotericin B in man.     

Liquid chromatographic determination of sotalol in plasma and urine employing solid-phase extraction and fluorescence detection

A liquid chromatographic method using a solid-phase extraction procedure for the quantification of sotalol in plasma and urine is described. Sotalol is eluted from an extraction column with ethyl acetate-acetonitrile (1:2) and, after separation by reversed-phase high-performance liquid chromatography on a mu Bondapak C18 column, is quantified by fluorescence detection at excitation and emission wavelengths of 240 and 310 nm, respectively. The method has been demonstrated to be linear over the concentration ranges 10-6000 ng/ml in plasma and 0.5-100 micrograms/ml in urine. Mean inter-assay accuracy of the method for plasma ranged from 93 to 100% and for urine from 102 to 114%; precision ranged from 0.5 to 1.6% for plasma over a concentration range of 200-4000 ng/ml and for urine from 0.7 to 2.0% at concentrations of 2-50 micrograms/ml. Mass spectrometry confirmed the presence of sotalol in isolated chromatographic fractions of plasma and urine extracts from subjects given sotalol orally.     

High-performance liquid chromatographic methods for the determination of the penems SCH 29482 and FCE 22101 in human serum and urine.

High-performance liquid chromatographic methods have been developed for the determination of two 6-(1-hydroxyethyl)penems, SCH 29482 (I) and FCE 22101 (II), in serum and urine. Serum samples were combined with an equal volume of methanol to remove proteins and, after centrifugation, an aliquot of the supernatant was analysed by ion-pair chromatography on a reversed-phase C18 column with hexadecyltrimethylammonium bromide as the ion-pairing agent. The compounds were detected by their ultraviolet absorbance at 305 nm for II and 322 nm for I. Urine samples were diluted, filtered and analysed by the same chromatographic procedure. At concentrations of 1-500 micrograms/ml of each compound, the within- and between-day precisions were 1.8-3.6 and 2.6-5.1%, respectively. The detection limit was 0.2 micrograms/ml for I and 0.3 micrograms/ml for II.     

Determination of vitamin U and its degradation products by high- performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method has been developed for the determination of vitamin U in tablets and capsules. Threonine was employed as the internal standard through the assay. The o-phthalaldehyde derivatives were prepared and then chromatographed isocratically on a reversed-phase C18 column. The optimum reaction time for both vitamin U and threonine at pH 10.5 is 5 min. Vitamin U and its major degradation product in the dosage forms, viz., methionine sulphoxide, were separated and quantified with a relative standard deviation of about 1%, using a fluorescence detector with excitation and emission wavelengths at 340 and 450 nm respectively. A linear relationship has been established between the peak area ratio of vitamin U/threonine and the concentration of vitamin U over the range of 2.5-50 micrograms/ml.     

Determination of (S)-(-)-cathinone and its metabolites (R,S)-(-)-norephedrine and (R,R)-(-)-norpseudoephedrine in urine by high-performance liquid chromatography with photodiode-array detection.

A high-performance liquid chromatographic (HPLC) procedure with photodiode-array detection (DAD) is described for the determination of (S)-(-)-cathinone (S-CA) and its metabolites (R,S)-(-)-norephedrine (R-NE) and (R,R)-(-)-norpseudoephedrine (R-NPE) in urine. Extraction and clean-up of 1-ml urine samples were performed on a cyano-bonded solid-phase column using (+/-)-amphetamine as internal standard. The concentrated extracts were separated on a 3-microns ODS-1 column with acetonitrile-water-phosphoric acid-hexylamine as the mobile phase. Peak detection was done at 192 nm. The detection limits for S-CA and R-NE/R-NPE in urine were 50 and 25 ng/ml, respectively. The differentiation of the enantiomers of cathinone and norephedrine was achieved by derivatization with (S)-(-)-1-phenylethyl isocyanate to the corresponding diastereomers followed by HPLC-DAD on a 5-microns normal-phase column. The R and S enantiomers of norpseudoephedrine were determined by gas chromatography-mass spectrometry after on-column derivatization with (S)-(-)-N-trifluoroacetylprolyl chloride. Following a single oral dose of 0.5 mg/kg of S-CA, the concentrations found in urine ranged from 0.2 to 3.8 micrograms/ml of S-CA, from 7.2 to 46.0 micrograms/ml of R-NE and from 0.5 to 2.5 micrograms/ml of R-NPE.     

Determination of glycyrrhizin in rabbit plasma by high-performance liquid chromatography with photodiode-array ultraviolet detection and its pharmacokinetics application.

A simple and sensitive high-performance liquid chromatographic method for the determination of glycyrrhizin in rabbit plasma has been developed. Up to 0.1 ml of plasma containing glycyrrhizin was deproteinized by acetonitrile, which contained an internal standard (indomethacin). The supernatant was injected onto a LiChrospher RP-18 column using a methanol-water-ammonia solution (80:20:0.1, v/v, pH 3.0-3.2, adjusted with perchloric acid) as the mobile phase and ultraviolet detection at 254 nm, followed by ultraviolet spectrum identification (between 200 and 380 nm) with a photodiode-array detector. The method is rapid, easily reproduced, selective and sensitive. It was applied to pharmacokinetic studies of glycyrrhizin in rabbit, after a 2 mg/kg intravenous administration. A biphasic phenomenon with a rapid distribution followed by a slower elimination phase was observed from the plasma concentration-time curve. Compartmental analysis yielded a two-compartment model.     

High-performance liquid chromatographic assay of cefotaxime, desacetylcefotaxime and ceftriaxone in rat plasma

A simple and selective high-performance liquid chromatographic method is described for the analysis of the cephalosporins cefotaxime (CXM), desacetylcefotaxime (DACXM) and ceftriaxone (CFX) in rat plasma. Plasma was deproteinized with methanol, and the supernatant was directly injected into the chromatograph and monitored at 254 nm. For determination of the unbound drugs, a centrifugal ultrafiltration method was employed. The calibration curves were linear (r = 0.999) from 2.5 to 500 micrograms/ml; the detection limits were 100 ng/ml for DACXM and 250 ng/ml for CXM and CFX. The method was not interfered with by other plasma components, nor by barbital sodium or caffeine, and has been applied to study the pharmacokinetics of the cephalosporins in rats.     

Determination of marker constituents in radix Glycyrrhizae and radix Notoginseng by near infrared spectroscopy

High-performance liquid chromatographic (HPLC) methods were developed for the determination of glycyrrhizin in radix Glycyrrhizae and ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf and Rg, in radix Notoginseng. These methods were used as reference methods for near-infrared (NIR) spectroscopy. Spectroscopic calibrations were developed for the determination of glycyrrhizin, the total content of ginsenosides and the individual major ginsenosides Rb1, Rd, Re and Rg1. Standard errors of cross validation (SECV) were 1.22 mg g(-1) for glycyrrhizin (concentration range 21.3-34.1 mg g(-1)) and 0.99 mg g(-1) for the sum of ginsenosides (concentration range 55.3-71.1 mg g(-1)). The corresponding coefficients of determination (R2) were 0.94 and 0.98, respectively. The SECVs were generally less than a factor of 2.5 of the repeatability standard deviation of the HPLC methods.     

Stereoselective determination of R(-)- and S(+)-MK-571, a leukotriene D4 antagonist, in human plasma by chiral high-performance liquid chromatography.

A stereoselective high-performance liquid chromatographic method that utilizes fluorescence detection was developed for the selective and sensitive quantification of R(-)- and S(+)-enantiomers of MK-571 (1), a potent and specific leukotriene D4 antagonist, in human plasma. Racemic 1 was isolated from the acidified plasma using solid-phase extraction and the resulting residue was successfully reacted with isobutyl chloroformate and R(+)-1-(1-naphthyl)ethylamine in triethylamine-acetonitrile medium to form the diastereomer of each enantiomer. A structural analogue of 1 was used as internal standard. The derivatized sample was dissolved in 1,1,2-trichlorotrifluoroethane and an aliquot was chromatographed on a (R)-urea chiral column using a mobile phase containing 89% triethylamine-pentane (3:1000, v/v), 10% 2-propanol, and 1% acetonitrile at a flow-rate of 1.5 ml/min. The fluorescence response (excitation wavelength, 350 nm; emission wavelength, 410 nm) was linear (r2 greater than 0.999) for concentrations of enantiomers of 1 from 0.05 micrograms/ml, the lowest quantitation limit, up to 2.5 micrograms/ml. Intra-day coefficients of variation at 0.05 microgram/ml were 2.4% for the R(-)-isomer and 2.0% for S(+)-isomer. The corresponding inter-day coefficients of variation for R(-)- and S(+)-1 were 2.6 and 3.6%, respectively. The utility of the methodology was established by analysis of plasma samples from male volunteers receiving single intravenous and oral doses of racemic 1.     

Determination of the new fluoroquinolone fleroxacin and its N-demethyl and N-oxide metabolites in plasma and urine by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method is described for the determination of the new fluoroquinolone fleroxacin and its metabolites in plasma and urine. Plasma samples are deproteinized with acetonitrile, and, after evaporation and reconstitution of the supernatant, samples are analysed on a reversed-phase column. The limit of quantification is 10-20 ng/ml for the parent drug and 10 ng/ml for the metabolites, using a 0.2-ml sample. Urine samples are diluted with the mobile phase. An aliquot is then injected directly onto the column. The limits of quantification are 1 micrograms/ml for the parent drug and 0.5 micrograms/ml for the metabolites, using a 0.1-ml sample. The method has been successfully applied to pharmacokinetic studies of human volunteers and patients.     

High-performance liquid chromatographic determination of iothalamic acid in human plasma and urine.

A simple, rapid and sensitive method for the determination of iothalamic acid (IA) in both plasma and urine is reported. After extraction with ethyl acetate, IA was determined by strong anion-exchange high-performance liquid chromatography with ultraviolet detection at 254 nm. The lower limit of detection was 0.5 micrograms/ml. The average recovery was 73 and 57% from plasma and urine, respectively. Linearity was found over the investigated concentration range (up to 500 micrograms/ml for plasma and up to 10.0 mg/ml for urine). The reproducibility of the technique was good (coefficient of variation less than 6%) as was the precision and accuracy (coefficient of variation less than 2.5%). No interference from endogenous substances or any of the common drugs tested was found.     

Simultaneous determination of ampicillin and sulbactam by liquid chromatography: post-column reaction with sodium hydroxide and sodium hypochlorite using an active hollow-fibre membrane reactor

A high-performance liquid chromatographic method has been developed for the simultaneous determination of ampicillin (ABPC) and sulbactam (SBT) in serum and urine. The method involves separation of ABPC and SBT from the background components of serum and urine on a C18 column, post-column reaction with sodium hydroxide and sodium hypochlorite using an active hollow-fibre membrane reactor, and detection at 270 nm. At ABPC and SBT concentrations of 10 and 5 micrograms/ml in urine and serum samples, the precisions (relative standard deviations) were 0.9-2.5% (n = 8). The detection limits were 20 and 5 ng for ABPC and SBT, respectively, at a signal-to-noise ratio of 3.     

Sensitive quantification of pseudoephedrine in human plasma and urine by high-performance liquid chromatography

An assay for the selective quantification of pseudoephedrine in human plasma and urine was developed using high-performance liquid chromatography with UV detection at 205 nm. Analyte and internal standard were extracted from alkaline plasma or urine into a mixture of n-hexane and diethyl ether, and the organic phase was back-extracted into dilute acid. The chromatographic system comprises microparticulate cyanopropyl-silica as stationary phase and a ternary solvent mixture with ion-pair reagents as mobile phase. Using 0.25 ml plasma, the lower limit of quantification was 25 ng/ml with excellent linearity up to 1000 ng/ml. In urine, the calibration ranged from 2.5 to 100 micrograms/ml. The selectivity of the method was demonstrated for several pharmaceuticals with similar structures. The validated method was applied to a pharmacokinetic study with a single oral dose of 100 mg of pseudoephedrine in two galenic formulations. Precision and accuracy data of the assay and calculated pharmacokinetic parameters are presented.     

Determination of cetirizine in human urine by high-performance liquid chromatography.

A high-performance liquid chromatographic method for the determination of the histamine H1-receptor antagonist cetirizine in human urine was developed. Cetirizine and the internal standard are extracted from acidified (pH 5) urine (0.5 ml) into chloroform and the organic layer is evaporated to dryness. The residue is chromatographed on a Spherisorb 5ODS-2 column using Pic A (5 mM aqueous tetrabutylammonium phosphate)-methanol-tetrahydrofuran (33:65:2, v/v) as the mobile phase with ultraviolet detection (230 nm). The calibration graph is linear from 0.1 to 10 micrograms/ml and using 0.5 ml of urine the detection limit is 20 ng/ml. The within-run relative standard deviation is less than 6% and the accuracy is within 10% of the theoretical value at concentrations between 0.1 and 10 micrograms/ml in urine. There is a good correlation (r = 0.99606) with a previously described capillary gas chromatographic assay.     

High-performance liquid chromatographic determination of cefonicid in human plasma and urine

A high-performance liquid chromatographic assay for determination of cefonicid concentrations in human plasma and urine samples has been developed using cefazolin as an internal standard. For the analysis of plasma samples two calibration curves were utilized covering the cefonicid concentration ranges of 0.05-1.0 microgram/ml and 1.0-50.0 micrograms/ml, respectively. Coefficients of variation of 7.4% or less were obtained for cefonicid concentrations of 0.05-50.0 micrograms/ml. Mean bias was +6.0% at 0.05 micrograms/ml cefonicid and between -2.1% and +1.6% for 1.0-50.0 micrograms/ml cefonicid. Plasma samples containing 30 ng/ml cefonicid could be well distinguished from blank plasma samples. Urine samples were analysed by using a calibration curve for cefonicid concentrations between 1.0 and 50.0 micrograms/ml. ranged from 8.6% at a cefonicid concentration of 1.0 microgram/ml to 0.5% at 50.0 micrograms/ml with a mean bias between -3.0% and +0.3%.     

Improvement of selectivity and sensitivity by column switching in the determination of glycyrrhizin and glycyrrhetic acid in human plasma by high-performance liquid chromatography

A sensitive method is described for the simultaneous determination of glycyrrhizin and glycyrrhetic acid in human plasma. Quantitation is by high-performance liquid chromatography using ion-pair chromatography on a reversed-phase column. Variable-wavelength ultraviolet detection is employed. For the extraction of both compounds from plasma, a new solid-phase ion-pair extraction procedure using octadecylsilane columns was developed. Because of the strong forces involved in the protein binding of glycyrrhizin, quantitative extraction of this compound from plasma was possible only after an alkaline pH shift. A considerable improvement in selectivity and sensitivity was obtained by automated column switching involving on-line preseparation of the solid-phase extract on a short precolumn and chromatographic analysis of a heart-cut from the precolumn eluate. The limit of detection of both glycyrrhizin and glycyrrhetic acid was 0.1 mg/l in 0.5 ml of plasma. From a preliminary study in human volunteers, it was concluded that glycyrrhetic acid rather than glycyrrhizin is preferred in a study in human volunteers to assess the zero effect level of glycyrrhizin.     

Rapid and specific method for the determination of vancomycin in plasma by high-performance liquid chromatography on an aminopropyl column

A high-performance liquid chromatographic method has been developed for the quantitative analysis of vancomycin in plasma. The method involves protein precipitation with acetonitrile, followed by normal-phase chromatography on an aminopropyl column. The clear supernatant was injected after centrifugation, and the eluent was monitored at 240 nm. No interference was found either with endogenous substances or with many currently used drugs, indicating a good selectivity for the procedure. The standard curve was linear between 0.1 and 100 micrograms/ml, and the detection limit was 0.01 microgram/ml of plasma. The mean intra- and inter-assay coefficients of variation were 2.4 and 4.0%, respectively, in the 10-50 micrograms/ml range. Application of the method to the study of vancomycin pharmacokinetics in a rabbit after a single intravenous dose is also reported.