On-line dialysis and weak cation-exchange enrichment of dialysate : Automated high-performance liquid chromatography of pholcodine in human plasma and whole blood

An automated method for the determination of pholcodine in plasma and whole blood is described. The technique combines dialysis and trace enrichment prior to high-performance liquid chromatography. Dialysis, trace enrichment on a weak cation-exchange column, separation on a cyano column and fluorescence detection was shown to be an extremely selective and sensitive method. The method has been used successfully in the analysis of real samples after administration of pholcodine. The automated method can be used, after minor modification, to determine other basic drugs in whole blood and plasma.     

Determination of intoplicine, a new antitumour drug, in human whole blood and plasma by normal-phase high-performance liquid chromatography with fluorescence detection

Intoplicine, a benzopyrido-indole derivative, is a novel anticancer agent currently under phase I clinical evaluation. A selective, sensitive normal-phase high-performance liquid chromatographic (HPLC) assay with fluorescence detection, suitable for the determination of intoplicine in human plasma and whole blood, is described. The sample pretreatment involves a protein precipitation step with 2-propanol. The reported assay was validated, and the stability of the analyte in plasma, in whole blood and in the extraction fluid was investigated. The method has been implemented in a pharmacokinetic phase I clinical trial with intoplicine given as a 24-h intravenous infusion.     

A precise method for the determination of whole blood and plasma sulfhydryl groups

A colorimetric method for the determination of total sulfhydryl content whole blood or plasma is presented. For whole blood, a mixture of fresh blood and 5,5′-dithiobis(2-nitrobenzoic acid) in diluted buffer is separated on a gel column into the yellow anion and the hemoglobin fraction. The plasma-DTNB mixture was read directly, and a correction for turbidity and/or color made after addition of N-ethylmaleimide. Results of a sample of human bloods are presented. The effects of several detergents yield high values of sulfhydryl in whole blood, which we believe to be artifactual. Studies of the stability of stored blood samples indicate that whole blood, but not plasma, can be kept refrigerated for several days without significant loss of sulfhydryl reaction.     

Improved micro-method for the high-performance liquid chromatographic determination of caffeine and paraxanthine in biological fluids

A high-performance liquid chromatographic procedure is reported for reproducibly and sensitively quantitating caffeine and its N-demethylated metabolite paraxanthine in micro-samples. A 5-μm reversed-phase radial compression column and 214-nm fixed wavelength ultraviolet detector were used to attain a sensitivity sufficient to quantitate these compounds at concentratios as low as 80 ng/ml using only 25 μl of sample. The assay is applicable to microliter samples of whole blood, serum, plasma, saliva, amniotic, cerebro-spinal and gastric fluids such as might be obtained in studies involving small animals or neonates. The utility of the assay is illustrated with caffeine and paraxanthine levels measured in several maternal and fetal fluids following constant-rate intravenous infusion of caffeine into a rabbit throughout pregnancy.     

Fully automated high-performance liquid chromatographic analysis of whole blood and plasma samples using on-line dialysis as sample preparation: Determination of oxytetracycline in bovine and salmon whole blood and plasma

A fully automated technique for high-performance liquid chromatographic analysis of whole blood and plasma is described. Samples are automatically injected into a dialyser where proteins and blood cells are removed. The dialysates are concentrated on a small column prior to analysis. This technique is used for the determination of oxytetracycline in whole blood and plasma. After dialysis oxytetracycline and the internal standard, tetracycline, are retained on a polystyrene enrichment column and subsequently separated on a polystyrene analytical column by ion-pair chromatography. Using ultraviolet detection 50 ng/ml can be detected. Validation showed good within-day and between-day accuracy and precision. Different oxytetracycline concentrations were found in plasma and whole blood. This difference varied between the species.     

High-performance liquid chromatographic method for determination of amodiaquine,chloroquine and their monodesethyl metabolites in biological samples

A high-performance liquid chromatographic method for determination of amodiaquine (AQ), desethylamodiaquine (DAQ), chloroquine (CQ) and desethylchloroquine (DCQ) in human whole blood, plasma and urine is reported. 4-(4-Dimethylamino-1-methylbutylamino)-7-chloroquinoline was used as internal standard. The drugs and the internal standard were extracted into di-isopropyl ether as bases and then re-extracted into an acidic aqueous phase with 0.1 M phosphate buffer at pH 4.0 for AQ samples and at pH 2.5 for CQ filter paper samples. A C(18) column was used and the mobile phase consisted of methanol-phosphate buffer (0.1 M, pH 3)-perchloric acid (250: 747.5:2.5, v/v). The absorbance of the drugs was monitored at 333 nm and no endogenous compound interfered at this wavelength. The limit of quantification in whole blood, plasma and urine was 100 nM for AQ and DAQ (sample size 100 microliter) as well as for CQ and DCQ in blood samples dried on filter paper. For 1000 microliter AQ and DAQ samples, the limit of quantification was 10 nM in all three biological fluids. The within-assay and between-assay coefficients of variations were always <10% at the limits of quantification. Plasma should be preferred for the determination of AQ and DAQ since use of whole blood may be associated with stability problems.     

The in vitro loss of penicillamine in plasma,albumin solutions,and whole blood: Implications for pharmacokinetic studies of penicillamine

The recent development of a high performance liquid chromatography assay method for the analysis of penicillamine in biological samples such as plasma, whole blood, and urine has provided a specific and sensitive assay method to aid in the study of penicillamine pharmacokinetics. Several investigators have reported measuring the plasma concentration of penicillamine. Some of these investigators have indicated that the plasma must be assayed immediately. However, such restrictions can limit the feasibility of a pharmacokinetic study. The results of this paper demonstrate the instability of penicillamine in plasma, albumin solutions, and whole blood. The rate of loss of penicillamine was shown to be influenced by the concentration of albumin. As a result of the significant loss of penicillamine over a short period of time, plasma or whole blood samples must be deproteinated immediately upon collection to avoid the loss of reduced penicillamine. Methods are presented for the preparation of biological samples so that the oxidation of penicillamine is prevented and the samples can be held for several days prior to analysis.     

Sensitive high-performance liquid chromatographic method with fluorescence detection for measurement of vinorelbine plasma concentrations

A high-performance liquid chromatographic (HPLC) method with fluorescence detection for the determination of vinorelbine in plasma is described. The technique was derived from that published by Debal for an assay of vinorelbine in cell culture medium. The modifications concern the preparation procedure for plasma samples (a two-step liquid-liquid extraction from plasma is desribed), optimization of the mobile phase composition, and use of a single C₁₈ column. These changes resulted in an improved sensitivity and reproducibility of the assay and led to its feasibility for clinical pharmacokinetic studies. The range of the assay is 2 to 1000 ng/ml.     

Determination of erythromycin in gastric juice and blood plasma by liquid chromatography and electrochemical detection

A liquid chromatographic method for determination of the antibiotic erythromycin in biological samples is described. Erythromycin and the internal standard, oleandomycin, were extracted from alkalinized samples with a mixture of 1-hexane and 2-butanol. After evaporation and reconstitution of the sample, separation was performed on a base-deactivated octadecylsilica column. The effects of pH in the mobile phase and of column temperature on the chromatographic performance were studied. Multiple and irregularly shaped peaks were obtained for some chromatographic systems, but by choosing appropriate conditions erythromycin could be eluted as a single symmetric peak. The absolute recovery was above 90% for erythromycin from blood plasma and above 85% from gastric juice. The limits of quantitation were 20 nM and 100 nM, respectively. Comparison of analytical results for a series of authentic samples with a microbiological assay showed excellent agreement.     

Improved method for the analysis of furosemide in plasma by high-performance liquid chromatography

Modifications of existing rapid high-performance liquid chromatographic procedures for the determination of furosemide in plasma were made in order to achieve greater sensitivity. To a small volume of plasma was added an internal standard structurally related to furosemide. Then, following previously described procedures, acetonitrile was added to precipitate the proteins and the clear supernatant was separated. However prior to injection of the supernatant the pH and composition of the sample were adjusted. This modification of the sample enabled an injection volume of up to 300 μl of the supernatant to be injected onto the chromatographic column. The effluent was monitored spectrofluorimetrically. A standard linear calibration curve with a mean precision of ± 4.4% was obtained for plasma samples containing 20–900 ng/ml of furosemide. Two structurally related compounds were used as internal standards in the furosemide assay.     

Improved and simplified liquid chromatography/electrospray ionization mass spectrometry method for the analysis of underivatized glucosamine in human plasma

Glucosamine is an amino monosaccharide reagent. It is difficult to assay using typical reversed-phase column due to the early elution, by optimizing the chromatographic conditions, especially the analytical column and the mobile phase composition, an improved analytical method was developed and validated, which offers rapid, sensitive and specific determination of glucosamine in human plasma. Following protein precipitation, the analyte and internal standard (valibose) were separated using an isocratic mobile phase on an Inertsil CN-3 column and detected by mass spectrometry in the multiple reaction monitoring mode using the respective precursor to product ion combinations of m/z 180/72 for glucosamine and m/z 252/198 for valibose. The chromatographic time was just 4.2 min for each sample, which made it possible to analyze more than 120 human plasma samples per day. The method exhibited a linear dynamic range of 4.00-4000 ng/mL for glucosamine in human plasma. The lower limit of quantification (LLOQ) was 4.00 ng/mL with a relative standard deviation of less than 10.9%. Acceptable precision and accuracy were obtained for the plasma concentrations over the standard curve range. By monitoring the two different MRM transitions, it was proved that no endogenous glucosamine was found in human plasma. The validated method has been successfully used to analyze human plasma samples for application in a bioequivalence study.     

Determination of propofol in rat whole blood and plasma by high-performance liquid chromatography

A simple, accurate and sensitive high-performance liquid chromatographic method was developed for the determination of propofol, an intravenous anaesthetic agent, in rat whole blood or plasma samples. The method is based on precipitation of the protein in the biological fluid sample and direct injection of the supernatant into an HPLC system involving a C₁₈ reversed-phase column using a methanol-water (70:30) mobile phase delivered at 1 ml/min. Propofol and the internal standard (4-tert.-octylphenol) were quantified using a fluorescence detector set at 276 nm (excitation) and 310 nm (emission). The analyte and internal standard had retention times of 6.3 and 10.5 min, respectively. The limit of quantification for propofol was 50 ng/ml using 100 μl of whole blood or plasma sample. Calibration curves were linear (r²=0.99) over a 1–10 μg/ml concentration range and intra- and inter-day precision were between 4–11%. The assay was applied to the determination of propofol whole blood pharmacokinetics and propofol whole blood to plasma distribution ratios in rats.     

Determination of 3'-amino-3'-deoxythymidine, a cytotoxic metabolite of 3'-azido-3'-deoxythymidine, in human plasma by ion-pair high-performance liquid chromatography

A sensitive high-performance liquid chromatographic assay has been developed to determine the levels of 3'-amino-3'-deoxy-thymidine (AMT), a cytotoxic metabolite of 3'-azido-3'-deoxy-thymidine (AZT, zidovudine), in human plasma. The sample pretreatment involved solid-phase extraction using cation-exchange extraction columns. Chromatography was carried out on a C₈ column, using a mobile phase of methanol—0.01 M ammonium acetate (pH 5)—0.25 M sodium dioctylsulfosuccinate (60:40:4, v/v/v) and ultraviolet detection at 265 nm. The method has been validated, and stability tests under various conditions have been performed. The lower limit of quantitation is 5 ng/ml (using 500-μl human plasma samples). The bioanalytical assay has been used for the determination of AMT in patients with AIDS who used AZT.     

Measurement of physiological concentrations of dapsone and its monoacetyl metabolite: a miniaturised assay for liquid or filter paper-absorbed samples

A modification of existing HPLC assay methods is described for the measurement of dapsone and monoacetyldapsone in 50-μl samples of plasma and whole blood. This method, in particular the use of small sample volumes dried onto filter paper strips, is applicable to multi-sample clinical and pharmacokinetic studies in children with malaria, who are often anaemic, and where sample volume must be kept to a minimum. Basified samples were extracted into 5 ml of ethyl acetate-tert.-butylmethyl ether (1:1, v/v), chromatographed on a μBondapaK C₁₈, 10 μm column with water-acetonitrile-glacial acetic acid (81:17.5:5, v/v) containing 2 g/l 1-octanesulphonic acid as the mobile phase and detected at 274 nm.     

一种改进的异硫氰酸胍法结合硅胶膜离心吸附柱提取孕妇外周血微量胎儿RNA的方法

孕妇外周血中存在胎儿RNA为无创性产前诊断提供了基础.但血液中富含RNA酶和微量胎儿RNA的特点,对从孕妇血浆中提取胎儿RNA带来困难. 我们以ε血红蛋白基因和胎盘特异表达基因4（PLAC4）mRNA作为研究对象,用改进的异硫氰酸胍法结合硅胶膜离心吸附柱法探索孕妇外周血中胎儿微量RNA的提取方法,获得满意效果. 30例孕妇和9例非孕妇外周血样品中总RNA经凝胶电泳测定显示3条带,分别为28S, 18S和 5.8S. 其28S条带亮度为18S亮度的2倍.总RNA质量浓度（A260／A280）为1.97 g/L,光密度比值(A260-A320）/（A280- A 320）为1.86. 30例孕妇外周血样本有7例提取到ε血红蛋白基因mRNA,ε血红蛋白基因 mRNA 的最小浓度为0.537 μg/mL,最大浓度为1.79 μg/mL,ε血红蛋白基因mRNA的浓度中位数为124 μg/mL. 30例孕妇外周血样本提取到PLAC4 基因mRNA,浓度最小值为2.105×103 copies/mL,最大值为12.760×103 copies/mL,而9例非孕妇中均未提取到（P＜0.01）,浓度中位数为6.612×103 copies/mL. 因此,改进的异硫氰酸胍法与硅胶膜离心吸附柱纯化法相结合,可有效抑制RNA降解,用于提取、纯化孕妇血液中微量胎儿RNA.     

A column-switching high-performance liquid chromatographic assay for a novel cytotoxic thioxanthone derivative (WIN 33377) in mouse plasma with toxicokinetic results from a mouse LD10 study

WIN 33377 (I) is a member of a novel class cytotoxic antitumor agents, 4-aminomethyl thioxanthane derivatives. A simple, rapid and reproducible method has been developed for the assay of I in mouse plasma using a high-performance liquid chromatographic method utilizing a column-switching technique. The method involves direct injection of buffered plasma to the extraction column for sample clean-up followed by switching onto an analytical column for analysis with UV detection at 256 nm. The method has demonstrated accuracy and precision over the range 10–2500 ng/ml using a 100-μl plasma sample with a minimum quantifiable level at 10 ng/ml. Stability of mouse plasma samples was demonstrated after storage for 4 weeks at −15 to −20°C, as was the ability of samples to be accurately quantified after a maximum of three freeze-thaw cycles. Recovery was greater than 87% for the compound and the internal standard. The assay was accurate and reproducible with measured values lying within the limits of defined acceptance criteria. The utility of the method was demonstrated by analyzing plasma samples obtained from mice dosed with I as part of a pre-clinical safety study intended to assist in the design of a pharmacokinetically guided dose escalation strategy.     

An improved radioenzymatic assay for plasma norepinephrine using purified phenylethanolamine N-methyltransferase

Radioenzymatic assays have been developed for norepinephrine (NE) using either catechol O-methyltransferase (COMT) or phenylethanolamine N-methyltransferase (PNMT). Assays using PNMT are specific for NE but have been considered less sensitive than the more complex assay procedures employing COMT. An improved purification procedure for bovine PNMT has permitted development of a NE assay with substantially improved sensitivity (less than 0.5 pg), reproducibility, and decreased manipulative effort. PNMT was purified by sequential pH 5.0 treatment and dialysis and by column chromatographic procedures using DEAE-Sephacel, Sephacryl S-200 and Phenyl Boronate-agarose. Recovery of PNMT activity through the purification scheme was 50% while blank recovery was less than 0.001%. Norepinephrine can be directly quantified in 25 microliters of human plasma and a seventy-tube assay can be routinely completed within 4 h. The capillary to venous plasma NE gradient was examined in eight normotensive male subjects. Capillary plasma NE (211 +/- 21.7 pg/ml) was significantly lower than venous plasma NE (367 +/- 32.7 pg/ml) in all subjects (p less than 0.005). This difference suggests the concentration of NE in capillary blood may be a unique indicator of sympathetic nervous system activity in vivo.     

Enantioselective and highly sensitive determination of carvedilol in human plasma and whole blood after administration of the racemate using normal-phase high-performance liquid chromatography

A highly sensitive HPLC method for enantioselective determination of carvedilol in human whole blood and plasma was developed. Carvedilol and S-carazolol as an internal standard extracted from whole blood or plasma were separated using an enantioselective separation column (Chiralpak AD column; 2.0 diameter x 250 mm) without any chiral derivatizations. The mobile phase was hexane:isopropanol:diethylamine (78:22:1, v/v). The excitation and emission wavelengths were set at 284 and 343 nm, respectively. The limits of quantification for the S(-)- and R(+)-carvedilol enantiomers in plasma and blood were both 0.5 ng/ml. Intra- and inter-day variations were less than 5.9%. As an application of the assay, concentrations of carvedilol enantiomer in plasma and blood samples from 15 patients treated with carvedilol for congestive heart failure were determined.     

Determination of metyrapone and the enantiomers of its chiral metabolite metyrapol in human plasma and urine using coupled achiral-chiral liquid chromatography

A coupled achiral-chiral liquid chromatographic assay has been developed to determine the concentrations of metyrapone and the enantiomers of its chiral metabolite metyrapol in plasma and urine. The chromatographic system consisted of a silica precolumn (75 × 4.6 mm I.D.) coupled in-line to a 250 × 4.6 mm I.D. column containing cellulose tris(4-methylbenzoate) coated on silica gel (Chiralcel OJ-CSP). When plasma samples were analyzed, the mobile phase was hexane-ethanol (92:8, v/v) modified with 0.1% diethylamine and when urine samples were analyzed the mobile phase was hexane-ethanol (94:6, v/v) modified with 0.2% diethylamine. Under these chromatographic conditions the chromatographic retentions [expressed as capacity factors (k′)] for metyrapone were k′ = 2.35 (plasma) and 2.52 (urine); for (−)-metyrapol k′ = 4.22 (plasma) and 4.62 (urine); for (+)-metyrapone k′ = 5.16 (plasma) and 5.86 (urine); enantioselectivities (α) were 1.09 (plasma) and 1.13 (urine). The assay has been validated for use in metabolic studies. The analyses of plasma and urine samples from one subject following oral administration of 750 mg of metyrapone indicated that the enzymatic reduction of myterapone by aldo-keto reductase was enantiospecific.     

Fast analysis using monolithic columns coupled with high-flow on-line extraction and electrospray mass spectrometric detection for the direct and simultaneous quantitation of multiple components in plasma

In this work, monolithic columns were used as a fast separation tool for multiple-component quantitative liquid chromatography-tandem mass spectrometry (LC-MS-MS) assays of drug candidates in biological fluids. A considerably reduced runtime was achieved while maintaining good chromatographic separations. This significantly improved separation speed demanded higher throughput on sample extraction. To this end, monolithic separations were coupled on-line with high-flow extraction, which allowed for the fast extraction and separation of samples containing multiple analytes. An evaluation of this system was performed using a mixture of fenfluramine, temazepam, oxazepam, and tamoxifen in plasma. A total cycle time of 1.2 min was achieved which included both sample extraction and subsequent monolithic column separation via column switching. A total of over 400 plasma samples were analyzed in less than 10 h. The sensitivity and responses were reproducible throughout the run. The system has been routinely used in the authors' laboratory for high-throughput quantitation of compounds in biological fluids in support of drug discovery programs. The assay for samples from a 9-in-1 dog pharmacokinetic study is shown as an example to demonstrate the capability of this system.