Yasuji Kasuya

High-performance liquid chromatographic determination of quercetin in human plasma and urine utilizing solid-phase extraction and ultraviolet detection

An HPLC method for determining quercetin in human plasma and urine is presented for application to the pharmacokinetic study of rutin. Isocratic reversed-phase HPLC was employed for the quantitative analysis by using kaempferol as an internal standard. Solid-phase extraction was performed on an Oasis HLB cartridge (>95% recovery). The HPLC assay was carried out using a Luna ODS-2 column (150 x 2.1 mm I.D., 5 microm particle size). The mobile phase was acetonitrile-10 mM ammonium acetate solution containing 0.3 mM EDTA-glacial acetic acid, 29:70:1 (v/v, pH 3.9) and 26:73:1 (v/v, pH 3.9) for the determination of plasma and urinary quercetin, respectively. The flow-rate was 0.3 ml/min and the detection wavelength was set at 370 nm. Calibration of the overall analytical procedure gave a linear signal (r>0.999) over a concentration range of 4-700 ng/ml of quercetin in plasma and 20-1000 ng/ml of quercetin in urine. The lower limit of quantification was approximately 7 ng/ml of quercetin in plasma and approximately 35 ng/ml in urine. The detection limit (defined at a signal-to-noise ratio of about 3) was approximately 0.35 ng/ml in plasma and urine. A preliminary experiment to investigate the plasma concentration and urinary excretion of quercetin after oral administration of 200 mg of rutin to a healthy volunteer demonstrated that the present method was suitable for determining quercetin in human plasma and urine.

Determination of rutin in human plasma by high-performance liquid chromatography utilizing solid-phase extraction and ultraviolet detection

An HPLC method for determining a flavonol glycoside, rutin, in human plasma is presented for application to the pharmacokinetic study. Isocratic reversed-phase HPLC was employed for the quantitative analysis by using kaempferol-3-rutinoside as an internal standard. Solid-phase extraction was performed on an Oasis MAX cartridge possessing reversed-phase and anion-exchange functions (recovery, approximately 80%). The HPLC assay was carried out using a Luna ODS-2 column (150 x 2.1 mm I.D., 5 microm particle size). The mobile phase was acetonitrile-10 mM ammonium acetate solution containing 0.3 mM EDTA-glacial acetic acid (16.5:82.5:1, v/v, pH 3.8). The flow-rate was 0.3 ml/min. The detection wavelength was set at 370 nm. Calibration of the overall analytical procedure gave a linear signal (r>0.9999) over a concentration range of 3-1,000 ng/ml of rutin in plasma. The lower limit of quantification was ca. 5 ng/ml of rutin in plasma. The detection limit (defined as signal-to-noise ratio of about 3) was approximately 0.75 ng/ml. A preliminary experiment to investigate the plasma concentration of rutin after oral administration of 500 mg of rutin to a healthy volunteer demonstrated that the present method was suitable for determining rutin in human plasma.

The pharmacokinetics of insulin after continuous subcutaneous infusion or bolus subcutaneous injection in diabetic patients.

Pharmacokinetic models of insulin were examined in order to describe a plasma concentration-time profile after subcutaneous (s.c.) administration of insulin to the patients with insulin-dependent diabetes mellitus (IDDM) or non-insulin-dependent diabetes mellitus (NIDDM). Diabetic subjects were restricted to those with fasting plasma insulin levels around the lowest limit for insulin assay (5 μU/ml). A one-compartment open model with first-order absorption and elimination was appropriate for estimating the plasma concentration-time profile of insulin injected or infused subcutaneously. In the case of continuous s.c. insulin infusion (CSII) for 1 h at the rate of 3 ml/h (2–3 U/ml), the absorption rate constant (Ka), elimination rate constant (Ke), and distribution volume (Vd) were 0.026 ± 0.001 min−1 (mean ± SEM; absorption half-life: 27 min), 0.013 ± 0.005 min−1 (elimination half-life: 53 min), and 1.99 ± 0.49 L/kg body wt, respectively. These values did not differ significantly from those generated by single bolus s.c. injection of undiluted insulin (40 U/ml). The calculated areas under the plasma insulin concentration-time curves from time zero to infinity ([AUC]∞0) did not differ after each mode of administration, while the [AUC] ∞0 after CSII was about 32% of that following intravenous bolus injection (P < 0.01). The following conclusions can be drawn from these results: (1) the plasma concentration-time profile of insulin after CSII or bolus s.c. injection can be analyzed by pharmacokinetic modeling, (2) the absorption kinetics of insulin did ot differ significantly between two modes of s.c. insulin administration in the patients with IDDM or NIDDM, and (3) the insulin after CSII or single bolus s.c. injection seems to be degraded at the s.c. site to the same extent.

Quantification of corticosteroids in human plasma by liquid chromatography-thermospray mass spectrometry using stable isotope dilution

Liquid chromatography-thermospray mass spectrometry (LC-TSP-MS) using isotope dilution was investigated for quantitative analysis of cortisol, cortisone, prednisolone and prednisone in human plasma. Complete separation attained by a LiChroCART Supersupher reversed-phase column and elution with 0.05 M ammonium formate-tetrahydrofuran-methanol (180:53:17, v/v/v) resulted in a significantly large isotope effect of the deuterium-labeled analogs on the HPLC behavior and caused difficulty in quantification. Reduction of the isotope effect on the retention times using 0.05 M ammonium formate-acetonitrile (65:35, v/v) permitted accurate quantification of cortisol and cortisone by the isotope dilution LC-TSP-MS, although separation between cortisol and prednisone was incomplete.

Determination of naringin and naringenin in human urine by high-performance liquid chromatography utilizing solid-phase extraction.

An HPLC method for determining a flavonoid naringin and its metabolite, naringenin, in human urine is presented for application to the pharmacokinetic study of naringin. Isocratic reversed-phase HPLC was employed for the quantitative analysis by using hesperidin for naringin or hesperetin for naringenin as internal standard and solid-phase extraction using a strong anion exchanger, Sep-Pak Accell QMA cartridge. The HPLC assay was carried out using an Inertsil ODS-2 column (250 x 4.6 mm I.D., 5 microm particle size). The mobile phases were acetonitrile-0.1 M ammonium acetate-acetic acid (18:81:1, v/v; pH 4.7) for naringin and acetonitrile-0.1 M ammonium acetate-triethylamine (25:75:0.05; v/v; pH 8.0) for naringenin. The flow-rate was 1.0 ml min(-1). The analyses were performed by monitoring the wavelength of maximum UV absorbance at 282 nm for naringin and at 324 nm for naringenin. The lower limits of quantification were ca. 25 ng/ml for naringin and naringenin with R.S.D. less than 10%. The lower limits of detection (defined as a signal-to-noise ratio of about 3) were approximately 5 ng for naringin and 1 ng for naringenin. A preliminary experiment to investigate the urinary excretion of naringin, naringenin and naringenin glucuronides after oral administration of 500 mg of naringin to a healthy volunteer demonstrated that the present method was suitable for determining naringin and naringenin in human urine.

Determination of naringin and naringenin in human plasma by high-performance liquid chromatography

An HPLC method for determining a flavonoid, naringin, and its metabolite, naringenin, in human plasma is presented for application to the pharmacokinetic study of naringin. Isocratic reversed-phase HPLC was employed for the quantitative analysis by using genistin (for naringin) or diadzein (for naringenin) as an internal standard and solid-phase extraction using s Sep-Pak t C18 cartridge. For the determination, HPLC was carried out using an Inertsil ODS-2 column (250 x 4.6 m I.D., 5 microns particle size). The mobile phases were acetonitrile-0.1 M ammonium acetate solution (20:80, v/v; pH 7.1) for naringin and acetonitrile-0.1 M ammonium acetate solution-acetic acid (30:69:1, v/v; pH 4.9) for naringenin. The flow-rate was 1 ml min-1. The analyses were performed by monitoring the wavelength of maximum UV absorbance at 280 nm for naringin and at 292 nm for naringenin. The detection limits on-column were about 0.2 ng for the two flavonoids.

Simultaneous determination of endogenous and 13C-labelled cortisols and cortisones in human plasma by stable isotope dilution mass spectrometry.

This study describes a capillary GC-MS method for the simultaneous determination of endogenous cortisol and cortisone and their 13C-labelled analogues, [1,2,4,19-13C4]cortisol (cortisol-13C4) and [1,2,4,19-13C4]cortisone (cortisone-13C4), in human plasma. [1,2,4,19-13C4,1,1,19,19,19-2H5]Cortisol (cortisol-13C4,2H5) and [1,2,4,19-13C4,1,1,19,19,19-2H5]cortisone (cortisone-13C4,2H5) were used as analytical internal standards. A double derivatization (bismethylenedioxy-pentafluoropropionate, BMD-PFP) with good GC behavior was employed for the GC-MS analysis of cortisol and cortisone. Quantitation was carried out by selected-ion monitoring of the molecular ions ([M]+*) of the BMD-PFP derivatives of cortisol and cortisone. The sensitivity limit of the present GC-MS-SIM method was found to be 150 pg per injection for cortisol (s/n=5.0) and 50 pg for cortisone (s/n=8.1). The within-day reproducibility in which the amounts of unlabelled and labelled cortisols and cortisones determined were in good agreement with the actual amounts added, the relative errors being less than 3.07%. The inter-assay coefficients of variation (C.V.) were less than 1.80% for unlabelled and labelled cortisols and cortisones.

Identification and quantification of 6β-hydroxydexamethasone as a major urinary metabolite of dexamethasone in man

Identification of 6 beta-hydroxydexamethasone as a major urinary metabolite of dexamethasone in man has been accomplished by nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. Mass fragmentographic measurements revealed that more than 30% of the intravenously or orally administered dexamethasone dose was excreted in the 24-h urine as 6 beta-hydroxydexamethasone, while only a small fraction of the dose was excreted as unchanged dexamethasone and its glucuronic acid conjugate.

Hydrolysis of conjugated steroids by the combined use of β-glucuronidase preparations from helix pomatia and ampullaria: determination of urinary cortisol and its metabolites

This study describes the enzymatic hydrolysis of urinary conjugates of cortisol, cortisone, tetrahydrocortisol, allotetrahydrocortisol, and tetrahydrocortisone with beta-glucuronidase preparations from Helix pomatia and Ampullaria. The objective of the present studies was to find optimal hydrolysis conditions for these conjugated steroids. Assay of the isolated steroids was carried out by GC-MS using deuterium-labeled compounds as internal standards. The allotetrahydrocortisol conjugate was clearly the hardest to hydrolyze with enzyme from Helix pomatia and required increased enzyme concentration and prolonged incubation. Hydrolysis of a urine sample for 2.0 h with the simultaneous use of 3400 units/ml Ampullaria and 5400 units/ml Helix pomatia enzymes in 0.5 M acetate buffer at 55 degrees C achieved more complete cleavage of the urinary conjugates of the five steroids examined. It is thus advantageous to use the Ampullaria and Helix pomatia enzymes in combination to obtain the highest yield in the urinary corticosteroid assay.

The use of deuterium-labeled cortisol for in vivo evaluation of renal 11β-HSD activity in man: urinary excretion of cortisol, cortisone and their A-ring reduced metabolites

This study describes a new approach using stable isotope methodology in evaluating 11beta-HSD activities in vivo based on urinary excretion of cortisol, cortisone, and their A-ring reduced metabolites. The method involved the measurement of deuterium-labeled cortisol and its deuterium-labeled metabolites by GC/MS simultaneously with endogenous cortisol, cortisone, and their A-ring reduced metabolites after oral administration of deuterium-labeled cortisol to normal human subjects. This stable isotope approach offered unique advantages in assessing the appropriateness of measuring unconjugated and total (unconjugated + conjugated) cortisol, cortisone, and their A-ring reduced metabolites in urine as indices of renal 11beta-HSD2 activity in man. Our results strongly support that the measurement of urinary unconjugated cortisol and cortisone is a significant advance in assessing 11beta-HSD2 activity.