Simultaneous determination of inulin and p-aminohippuric acid in plasma and urine by reversed-phase high-performance liquid chromatography

A simple, accurate and sensitive high-performance liquid chromatographic method with UV detection was carried out to measure simultaneously plasma and urine concentrations of both p-aminohippuric acid and inulin. Following a simplified acid hydrolysis of the sample, the separation was carried out in 4 min using a C18 reversed-phase column with a flow-rate of 1 ml/min, and monitoring the absorbance at 280 nm. Within the investigated concentration ranges of inulin (0.1-3.2 mg/ml) and p-aminohippuric acid (0.0097-0.3 mg/ml), good linearity (r>0.99) was obtained. Within-run RSD ranged from 2.9 to 6.1% and between-run RSD ranged from 6.4 to 10%. Analytical recoveries were 101-112%, with little differences between plasma and urine samples. The detection limit was 1 microg/ml for all the analytes studied. This method might be ideal for renal function studies where a rapid and reproducible assessment of both renal glomerular filtration rate and blood flow-rate is required.     

Automated quantitative determination of the new renin inhibitor CGP 60536 by high-performance liquid chromatography

A fully automated high-performance liquid chromatography method with fluorescence detection for the determination of the renin inhibitor CGP 60536 in animal and human plasma and urine has been developed and validated. After addition of an internal standard, the compounds were automatically extracted from 400 microl of plasma or urine with methyl alcohol-acetic acid (99:1, v/v) on 100-mg Bond-Elut CN cartridges using the Gilson ASPEC system. The on-line chromatographic separation was performed on a LiChrospher 100 RP8 5-microm particle size packed analytical column (25x0.4 cm I.D.). The mobile phase consisted of acetonitrile-0.01 M potassium dihydrogenphosphate (65:35, v/v) at a flow-rate of 0.8 ml/min. The analytes were detected using a fluorescence detector at excitation and emission wavelengths of 280 and 330 nm, respectively. The limit of quantitation was established at 4.5 ng/ml in plasma (accuracy 106% and precision 1%), and 9.0 ng/ml in urine (accuracy 101% and precision 13%). The method was applied to the investigation of the pharmacokinetics of CGP 60536.     

Analysis of aplidine (dehydrodidemnin B), a new marine-derived depsipeptide, in rat biological fluids by liquid chromatography-tandem mass spectrometry.

Aplidine (dehydrodidemnin B) is a new marine-derived depsipeptide with a powerful cytotoxic activity, which is under early clinical investigation in Europe and in the US. In order to investigate the pharmacokinetic properties of this novel drug, an HPLC-tandem mass spectrometry method was developed for the determination of aplidine in biological samples. Didemnin B, a hydroxy analogue, was used as internal standard. After protein precipitation with acetonitrile and extraction with chloroform, aplidine was chromatographed with a RP octadecylsilica column using a water-acetonitrile linear gradient in the presence of formic acid at the flow-rate of 500 microliters/min. The method was linear over a 5-100 ng/ml range (LOD = 0.5 ng/ml) in plasma and over a 1.25-125 ng/ml range (LOD = 0.2 ng/ml) in urine with precision and accuracy below 14.0%. The intra- and inter-day precision and accuracy were below 12.5%. The extraction procedure recoveries for aplidine and didemnin B were 69% and 68%, respectively in plasma and 91% and 87%, respectively in urine. Differences in linearity, LOQ, LOD and recoveries between plasma and urine samples seem to be matrix-dependent. The applicability of the method was tested by measuring aplidine in rat plasma and urine after intravenous treatment.     

Development of a high-performance liquid chromatographic method to determine the concentration of karenitecin, a novel highly lipophilic camptothecin derivative, in human plasma and urine

Karenitecin is a novel, highly lipophilic camptothecin derivative with potent anticancer potential. We have developed a sensitive high-performance liquid chromatographic method for the determination of karenitecin concentration in human plasma and urine. Karenitecin was isolated from human plasma and urine using solid-phase extraction. Separation was achieved by gradient elution, using a water and acetonitrile mobile phase, on an ODS analytical column. Karenitecin was detected using fluorescence detection at excitation and emission wavelengths of 370 and 490 nm, respectively. Retention time for karenitecin was 16.2 +/- 0.5 min and 8.0 +/- 0.2 min for camptothecin, the internal standard. The karenitecin peak was baseline resolved, with the nearest peak at 3.1 min distance. Using normal volunteer plasma and urine from multiple individuals, as well as samples from the 50 patients analyzed to date, no interfering peaks were detected. Inter- and intra-day coefficients of variance were <4.4 and 7.1% for plasma and <4.9 and 11.6% for urine. Assay precision, based on an extracted karenitecin standard plasma sample of 2.5 ng/ml, was +4.46% with a mean accuracy of 92.4%. For extracted karenitecin standard urine samples of 2.5 ng/ml assay precision was +2.35% with a mean accuracy of 99.5%. The mean recovery of karenitecin, at plasma concentrations of 1.0 and 50 ng/ml, was 81.9 and 87.8% respectively. In urine, at concentrations of 1.5 and 50 ng/ml, the mean recoveries were 90.3 and 78.4% respectively. The lower limit of detection (LLD) for karenitecin was 0.5 ng/ml in plasma and 1.0 ng/ml in urine. The lower limit of quantification (LLQ) for karenitecin was 1 ng/ml and 1.5 ng/ml for plasma and urine, respectively. Stability studies indicate that when frozen at -70 degrees C, karenitecin is stable in human plasma for up to 3 months and in human urine for up to 1 month. This method is useful for the quantification of karenitecin in plasma and urine samples for clinical pharmacology studies in patients receiving this agent in clinical trials.     

Determination of 5-hydroxy-N-methyl-2-pyrrolidone and 2-hydroxy-N-methylsuccinimide in human plasma and urine using liquid chromatography-electrospray tandem mass spectrometry

A method for simultaneous determination of 5-hydroxy-N-methyl-2-pyrrolidone (5-HNMP) and 2-hydroxy-N-methylsuccinimide (2-HMSI) was developed. These compounds are metabolites from N-methyl-2-pyrrolidone (NMP), a powerful and widely used organic solvent. 5-HNMP and 2-HMSI were purified from plasma and urine by solid-phase extraction using Isolute ENV+ columns, and analysed by liquid chromatography coupled to a mass spectrometer fitted with an atmospheric pressure turbo ion spray ionisation interface in the positive ion mode. The method was validated for plasma and urine concentrations from 0.12 to 25 microg/ml. The recoveries for 5-HNMP and 2-HMSI in plasma were 99 and 98%, respectively, and in urine 111 and 106%, respectively. For 5-HNMP and 2-HMSI, the within-day precision in plasma was 1-4 and 3-6%, respectively, and in urine 2-12 and 3-10%, respectively. The corresponding data for the between-day precision was 5 and 3-6%, respectively, and 4-6 and 7-8%, respectively. The detection limit for 5-HNMP was 4 ng/ml in plasma and 120 ng/ml in urine. For 2-HMSI, it was 5 ng/ml in plasma and 85 ng/ml in urine. The method is applicable for analysis of plasma and urine samples from workers exposed to NMP.     

Determination of a chemopreventive agent,Oltipraz, in rat plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the determination of a chemopreventive agent, Oltipraz, in rat plasma and urine. The sample preparation was simple; 2 volumes of acetonitrile were added to deproteinize the biological sample. A 50-microl aliquot of the supernatant was injected onto a C18 reversed-phase column. The mobile phase, acetonitrile : 0.5 mM ammonium acetate (55: 45, v/v for rat plasma and 45 : 55, v/v for rat urine), was run at a flow-rate of 1.5 ml/min. The column effluent was monitored using an ultraviolet detector set at 305 nm. The retention times for Oltipraz in rat plasma and urine were approximately 5.8 and 8.6 min, respectively. The detection limits of Oltipraz in rat plasma and urine were 20 and 50 ng/ml, respectively. The coefficients of variation of the assay (within-day and between-day) were generally low (below 4.65%) in concentration ranges from 0.02 (0.05) to 10 microg/ml for rat plasma and urine. No interference from endogenous substances was found.     

Narrowbore high-performance liquid chromatography for the simultaneous determination of sildenafil and its metabolite UK-103,320 in human plasma using column switching

A fully automated narrowbore high-performance liquid chromatography method with column switching was developed for the simultaneous determination of sildenafil and its active metabolite UK-103,320 in human plasma samples without pre-purification. Diluted plasma sample (100 microl) was directly introduced onto a Capcell Pak MF Ph-1 column (20x4 mm I.D.) where primary separation occurred to remove proteins and concentrate target substances using 15% acetonitrile in 20 mM phosphate solution (pH 7). The drug molecules eluted from the MF Ph-1 column were focused in an intermediate column (35x2 mm I.D.) by a valve switching step. The substances enriched in the intermediate column were eluted and separated on a phenyl-hexyl column (100x2 mm I.D.) using 36% acetonitrile in 10 mM phosphate solution (pH 4.5) when the valve status was switched back. The method showed excellent sensitivity (detection limit of 10 ng/ml), good precision (RSD < or = 2.3%) and accuracy (bias: +/-2.0%) and speed (total analysis time 17 min). The response was linear (r2 > or = 0.999) over the concentration range 10-1000 ng/ml.     

Highly sensitive analysis of the antifolate pemetrexed sodium, a new cancer agent, in human plasma and urine by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatography method was developed and validated for the quantitation of pemetrexed (LY231514, ALIMTA) in human urine and plasma. Plasma samples were spiked with the internal standard lometrexol and extracted using Certify II columns. Pemetrexed was assayed in diluted urine by an external calibration method. A C8 column was used for the separation of analytes with a mobile phase composed of sodium formate buffer and acetonitrile. Between- and within-day precision and accuracy were acceptable down to the limit of quantitation of 5 ng/ml in plasma. This method was used successfully for an investigation of the disposition of pemetrexed in patients receiving 500 mg/m2 as a 10-min infusion.     

Spectrofluorimetric estimation in biological fluids of a new beta-blockader: atenolol. Application to its pharmacokinetic study (author's transl)]

The authors describe a simple method of fluorimetric estimation of atenolol, applicable to blood and urine, sufficiently sensitive to permit a pharmacokinetic study. After administration of a dose of 200 mg by the oral route to hypertensive subjects, one may observe a maximal plasma concentration 3 hours after the dose average value 3.91 +/- 0.71 mumol/l (1.04 +/- 0.19 mg/l). The apparent half life of elimination of phase beta is 14.1 +/- 4.9 h, values comparable with those calculated from urinary data 14.6 +/- 2.0 h. The renal clearance was 140 +/- 32 ml/min; 54 +/- 14% of the administered dose are excreted in the urine. After administration by the venous route, urinary excretion represents 96% of the dose administered.     

Separation of carvedilol enantiomers in very small volumes of human plasma by capillary electrophoresis with laser-induced fluorescence

A sensitive capillary electrophoretic method for the determination of carvedilol enantiomers in 100 microl of human plasma has been developed and validated. Carvedilol and the internal standard carazolol are isolated from plasma samples by liquid-liquid extraction using diethylether. A sensitive and selective detection is provided by helium-cadmium laser-induced fluorescence. The total analysis time is 17.5 min, about 30 min are needed for the sample preparation. The linearity of the assay ranges from 1.56 to 50 ng/ml per carvedilol enantiomer. The limits of quantification (LOQ) for the carvedilol enantiomers in 100 microl of human plasma are 1.56 ng/ml. The inter-day accuracy for R-carvedilol is between 95.8 and 103% (104% at LOQ) and for S-carvedilol between 97.1 and 103% (107% at LOQ); the inter-day precision values are between 3.81 and 8.64% (10.9% at LOQ) and between 5.47 and 7.86% (7.91% at LOQ) for R- and S-carvedilol, respectively. The small sample volume needed is especially advantageous for the application in clinical studies in pediatric patients. As an application of the assay concentration/time profiles of the carvedilol enantiomers in a 5-year-old patient receiving a test dose of 0.09 mg/kg carvedilol are reported.     

Improved highly sensitive method for determination of nicotine and cotinine in human plasma by high-performance liquid chromatography

A highly sensitive and reliable method for the determination of nicotine and its metabolite cotinine in human plasma by high-performance liquid chromatography was developed. Nicotine and cotinine were extracted from alkalinized plasma with dichloromethane and the volatility of nicotine was prevented by the addition of conc. HCl to the organic solvent during evaporation. The sensitivity of quantification at 260 nm absorption was improved by using a noise-base clean Uni-3 to 0.2 ng/ml nicotine and 1.0 ng/ml cotinine. The method was validated over linear ranges of 0.2-25.0 ng/ml for nicotine and 1.0-80.0 ng/ml for cotinine. The intra-day precision and accuracy were < or = 15.9% relative standard variation (RSD) and 89.9-103.5% for nicotine and < or = 8.0% RSD and 98.7-103.0% for cotinine. The inter-day precision and accuracy were < or = 17.0% RSD and 94.2-100.9% for nicotine and < or = 8.2% RSD and 98.0-105.1% for cotinine.     

Determination of ginsenoside Rg3 in plasma by solid-phase extraction and high-performance liquid chromatography for pharmacokinetic study.

A method using high-performance liquid chromatography (HPLC) and solid-phase extraction (SPE) is described for the determination of ginsenoside Rg3 in human plasma. A 2.5-ml volume of plasma was mixed with 2.5 ml 60% methanol aqueous solution, and centrifuged at 1100 g for 10 min, the supernatant fluid was further purified by SPE with 200 mg/5 ml 40 microns octadecyl silica and separation was obtained using a reversed-phase column under isocratic conditions with ultraviolet absorbance detection. The intra- and inter-day precision, determined as relative standard deviations, were less than 5.0%, and method recovery was more than 97%. The lower limit of quantitation, based on standards with acceptable RSDs, was 2.5 ng/ml. No endogenous compounds were found to interfere with analyte. A good linear relationship with a regression coefficient of 0.9999 in the range of 2.5 to 200 ng/ml was observed. This method has been demonstrated to be suitable for pharmacokinetic studies in humans. Method development for determination of drug with low UV absorption by SPE and HPLC is also discussed.     

Direct analysis of platinum in plasma and urine by electrothermal atomic absorption spectrophotometry

An improved technique is described for analysis of platinum (Pt) concentrations in plasma and urine by electrothermal atomic absorption spectrophotometry (EAAS). The method is intended for use in therapeutic monitoring of cancer patients treated with platinum-containing antitumor drugs. Samples (0.1 ml) of plasma, urine, or Pt standards are diluted to two ml with a matrix solution that contains diammonium edetate, ammonium dihydrogen phosphate, ammonium hydroxide, and octoxynol detergent. Concentrations of Pt in the diluted samples are determined directly by EAAS analysis with Zeeman background correction. Standard additions are unnecessary; Pt concentrations are read from a calibration chart of peak heights, which is linear up to 1.6 mg per liter. The detection limit is 0.02 mg of Pt per liter. Day-to-day precision (coefficient of variation, based on 21 consecutive runs) ranges from 4.2 to 11.7 percent, depending upon the Pt concentration in the plasma and urine specimens. Recovery of Pt added to plasma and urine specimens averages 103 +/- 8 and 99 +/- 6 percent, respectively. Concentrations of Pt are stable in plasma and urine specimens stored at 4 degrees C or -20 degrees C for four weeks. Analyses of Pt concentrations in serial plasma and urine specimens from cancer patients receiving cisplatin chemotherapy demonstrate the clinical utility of the technique.     

A high-performance liquid chromatography assay with ultraviolet detection for olanzapine in human plasma and urine

Olanzapine is a commonly used atypical antipsychotic medication for which therapeutic drug monitoring has been proposed as clinically useful. A sensitive method was developed for the determination of olanzapine concentrations in plasma and urine by high-performance liquid chromatography with low-wavelength ultraviolet absorption detection (214 nm). A single-step liquid-liquid extraction procedure using heptane-iso-amyl alcohol (97.5:2.5 v/v) was employed to recover olanzapine and the internal standard (a 2-ethylated olanzapine derivative) from the biological matrices which were adjusted to pH 10 with 1 M carbonate buffer. Detector response was linear from 1-5000 ng (r2>0.98). The limit of detection of the assay (signal:noise=3:1) and the lower limit of quantitation were 0.75 ng and 1 ng/ml of olanzapine, respectively. Interday variation for olanzapine 50 ng/ml in plasma and urine was 5.2% and 7.1% (n=5), respectively, and 9.5 and 12.3% at 1 ng/ml (n=5). Intraday variation for olanzapine 50 ng/ml in plasma and urine was 8.1% and 9.6% (n=15), respectively, and 14.2 and 17.1% at 1 ng/ml (n=15). The recoveries of olanzapine (50 ng/ml) and the internal standard were 83 +/- 6 and 92 +/- 6% in plasma, respectively, and 79 +/- 7 and 89 +/- 7% in urine, respectively. Accuracy was 96% and 93% at 50 and 1 ng/ml, respectively. The applicability of the assay was demonstrated by determining plasma concentrations of olanzapine in a healthy male volunteer for 48 h following a single oral dose of 5 mg olanzapine. This method is suitable for studying olanzapine disposition in single or multiple-dose pharmacokinetic studies.     

Uric acid excretion by the rat kidney.

The renal excretion of uric acid was studied in nondiuretic (ND) male Wistar rats and in the same animals subsequently made diuretic (D) by the infusion of hypertonic saline. Clearances of endogenous urate and of inulin, determined chemically, were compared with the simultaneous clearance of 14C infused as [6(-14)C]urate or [2(-14)C]urate. In rats infused with [6(-14)C]urate the isotope/inulin clearance ratios were 0.29 +/- 0.09 (ND) and 0.31 +/- 0.11 (D) ml/min; the simultaneous urate (chemical)/inulin ratios were 0.21 +/- 0.07 (ND) and 0.24 +/- 0.08 (D) ml/min. In rats infused with [2(-14)C]urate the isotope/inulin clearance ratios were 1.02 +/- 0.5 (ND) and 1.13 +/- 0.9 ml/min (D); the simultaneous urate (chemical)/inulin clearance ratios were much lower-0.19 +/- 0.09 (ND) and 0.32 +/- 0.19 (D) ml/min. Thin-layer chromatography of urine after [6(-14)C]urate inl uric acid. In contrast, a similar analysis of urinary radioactivity after [2(-14)C]urate infusion revealed that more than 70% of the 14C was excreted as allantoin and not as uric acid.     

Determination of (E)-5-(2-bromovinyl)-2'-deoxyuridine in plasma and urine by capillary electrophoresis.

(E)-5-(2-Bromovinyl)-2'-deoxyuridine is an antiviral drug used for treatment of infections with Herpes simplex virus type 1 as well as Varicella zoster virus. Two fast methods for the determination of the drug and its metabolite in plasma and urine by capillary electrophoresis have been developed. The plasma method can be used for measurement of total as well as unbound drug and metabolite. Plasma and urine samples are prepared for measuring by liquid/liquid extraction resulting in a limit of quantification of 40 ng/ml for total and 10 ng/ml for free BVdU in plasma and 170 ng/ml in urine. Inter- as well as intra-day precision were found to be better than 10% and both methods have been used for drug monitoring of patients.     

The source of inulin in samples of vasa recta blood

Vasa recta blood was sampled from the exposed renal papillae of 11 Munich-Wistar rats (Group I). The mean vasa recta plasma-to-systemic plasma (VR/P) inulin concentration ratio was 5.4±1.2 (SE), which is significantly higher than the mean VR/P protein ratio previously reported (1.74 in descending vasa recta and 1.38 in acending vasa recta). Even if glomerular and vasa recta capillaries are as impermeable to inulin as to protein, VR/P inulin concentration ratios should not exceed VR/P protein concentration ratios, unless inulin is added to vasa recta from an extracapillary source. To determine if inulin can permeate the epithelial lining of the papilla, we bathed exposed papillae with concentrated inulin solutions (5 rad 10 g/ 100 ml). In 7 rats (Group II) the inulin concentration in 23 VR plasma samples ranged from 0 to 492 mg/100 ml, exhibiting large variations within individual rats. A third group of 12 rats, infused intravenously with inulin, exhibited the same pattern of variability. We interpret these data as evidence for occasional contamination of vasa recta blood by urine in adjacent collecting tubules or bathing the papilla. A preliminary report of this investigation was presented to the Western Society for Clinical Research, Carmel, California, February, 1976.     

Development of a radioimmunoassay for measuring gonadotrophin releasing hormone in patients receiving treatment.

A radioimmunoassay for the measurement of gonadotrophin releasing hormone (GnRH) in plasma and urine using readily available reagents was developed. The GnRH assay showed good precision, recovery, and parallelism over a wide range of GnRH concentrations with a sensitivity of 15 pg/ml. The assay was compared with a commercially available kit (Buhlmann Laboratories). Although the Buhlmann kit showed acceptable precision, recovery, sensitivity, and correlation with the developed GnRH assay for plasma samples, lack of parallelism of serially diluted plasma and urine samples was consistently observed, together with a poor correlation with the developed GnRH assay for urine, suggesting a matrix effect with the Buhlmann kit. The developed assay is suitable for measuring GnRH in samples obtained from patients receiving pulsatile infusions of GnRH. In contrast, the commercially available Buhlmann kit was unsuitable for measuring plasma GnRH as the kit had a top standard of only 160 pg/ml, well below the peak plasma concentration. It would not be possible to dilute samples for analysis because of the lack of parallelism of diluted samples compared with standards obtained with the Buhlmann assay.     

Simple and sensitive high-performance liquid chromatography method for the determination of docetaxel in human plasma or urine

Several methods for quantification of docetaxel have been described mainly using HPLC. We have developed a new isocratic HPLC method that is as sensitive and simpler than previous methods, and applicable to use in clinical pharmacokinetic analysis. Plasma samples are spiked with paclitaxel as internal standard and extracted manually on activated cyanopropyl end-capped solid-phase extraction columns followed by isocratic reversed-phase HPLC and UV detection at 227 nm. Using this system, the retention times for docetaxel and paclitaxel are 8.5 min and 10.5 min, respectively, with good resolution and without any interference from endogenous plasma constituents or docetaxel metabolites at these retention times. The total run time needed is only 13 min. The lower limit of quantification is 5 ng/ml using 1 ml of plasma. The validated quantitation range of the method is 5-1000 ng/ml with RSDs < or = 10%, but plasma concentrations up to 5000 ng/ml can be accurately measured using smaller aliquots. This method is also suitable for the determination of docetaxel in urine samples under the same conditions. The method has been used to assess the pharmacokinetics of docetaxel during a phase I/II study of docetaxel in combination with epirubicin and cyclophosphamide in patients with advanced cancer.     

Determination of 3'-azido-2',3'-dideoxyuridine in maternal plasma, amniotic fluid, fetal and placental tissues by high-performance liquid chromatography

3'-Azido-2',3'-dideoxyuridine (AZDU, Azddu, CS-87) is a nucleoside analog of 3'-azido-3'-deoxythymidine (zidovudine, AZT) that has been shown to inhibit human immunodeficiency virus (HIV-1). AZDU is a potential candidate for treatment of pregnant mothers to prevent prenatal transmission of HIV/AIDS to their unborn children. A rapid and efficient high-performance liquid chromatography (HPLC) method for the determination of AZDU concentrations in rat maternal plasma, amniotic fluid, placental and fetal tissue samples has been developed and validated. Tissue samples were homogenized in distilled water, protein precipitated and extracted using a C-18 solid-phase extraction (SPE) method prior to analysis. Plasma and amniotic fluid samples were protein precipitated with 2 M perchloric acid prior to analysis. Baseline resolution was achieved using a 4.5% acetonitrile in 40 mM sodium acetate (pH 7) buffer mobile phase for amniotic fluid, placenta and fetus samples and with a 5.5% acetonitrile in buffer solution for plasma at flow-rates of 2.0 ml/min. The HPLC system consists of a Hypersil ODS column (150x4.6 mm) with a Nova-Pak C-18 guard column with detection at 263 nm. The method yields retention times of 6.2 and 12.2 min for AZDU and AZT in plasma and 8.3 and 17.6 min for AZDU and AZT in amniotic fluid, fetal and placental tissues. Limits of detection ranged from 0.01 to 0.075 microg/ml. Recoveries ranged from 81 to 96% for AZDU and from 82 to 96% for AZT in the different matrices. Intra-day (n=6) and inter-day (n=9) precision (% RSD) and accuracy (% Error) ranged from 1.48 to 6.25% and from 0.50 to 10.07%, respectively.