Yasuhiko Higashi

HPLC Analysis of Bucillamine by Derivatization with N ‐(1‐Pyrenyl)Maleimide in Human Blood

Abstract Bucillamine, N‐(2‐mercapto‐2‐methylpropionyl)‐L‐cysteine, is a novel disease‐modifying anti‐rheumatic drug containing two sulfhydryl groups. We investigated high performance liquid chromatographic (HPLC) analysis of bucillamine by derivatization with N‐(1‐pyrenyl)maleimide (NPM) in human blood. Human blood samples were immediately mixed with NPM at room temperature for 15 min and injected into an HPLC. The linearity was displayed for bucillamine concentrations ranging from 3 to 3000 nM (r = 0.999). The lower limit of detection of bucillamine derivative was 2.5 nM. Bucillamine derivative remained stable at −4°C for at least 2 weeks with less than 5% variation. The coefficients of variation for intra‐day and inter‐day assays were less than 4.2% and 5.3%, respectively. No endogenous and exogenous compounds (L‐cysteine, glutathione, N‐acetyl‐L‐cysteine, captopril, and D‐penicillamine), possessing a sulfhydryl group in these structures, were found to interfere with the peak of the bucillamine derivative. These results indicate that HPLC assay of bucillamine by derivatization with NPM is rapid, sensitive, and reproducible for determining bucillamine in human blood. This method may be applied to pharmacokinetic studies of bucillamine in humans.

Determination of Fluoxetine and Norfluoxetine in Human Serum and Urine by HPLC Using a Cholester Column with Fluorescence Detection

Abstract Fluoxetine (FLX), a selective serotonin reuptake inhibitor, is mainly demethylated to norfluoxetine (NFLX). In this study, FLX and NFLX levels in human serum and urine (each 100 µL) were simultaneously analyzed by HPLC fluorescence detection on a Cholester column after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). After basic extraction of the samples into pentane, derivatization with NBD-F was conducted in borate buffer (pH 8.5) at 70°C for 2 min. Protriptyline was utilized as an internal standard. The regression equations for FLX hydrochloride and NFLX hydrochloride in human serum showed good linearity in the range of 0.01–0.5 µg/mL with the detection limit of 0.005 µg/mL, and in the range of 0.005–0.5 µg/mL with the detection limit of 0.002 µg/mL, respectively. The corresponding values for human urine were 0.1–0.5 µg/mL with the detection limit of 0.06 µg/mL, and 0.1–0.5 µg/mL with the detection limit of 0.04 µg/mL, respectively. The coefficients of variation were less than 15.7% with good recovery. Our method is useful for a simple and sensitive determination of FLX and NFLX in human serum and urine using a sample volume as small as 100 µL, and should be suitable for therapeutic drug monitoring.

HPLC-UV ANALYSIS OF EUGENOL IN CLOVE AND CINNAMON OILS AFTER PRE-COLUMN DERIVATIZATION WITH 4-FLUORO-7-NITRO-2,1,3-BENZOXADIAZOLE

Eugenol is one of the flavor constituents in various medicinally used plant oils, such as clove and cinnamon oils, which are widely used as flavoring agents in foods and beverages. In this study, the levels of eugenol in clove and cinnamon oils were analyzed by HPLC-UV (380 nm) after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). A standard curve was obtained after derivatization with NBD-F in borate buffer (pH 8.5) at 40°C for 4 min. The retention time of NBD-eugenol was 12.1 min. The calibration plot was linear in the range of 0.2 to 5 μg/mL with an r2 value of 0.9976, and the lower limit of detection was 0.04 μg/mL (at a signal-to-noise ratio of 3:1). The coefficient of variation was less than 6.7%. It was found that the content of eugenol in clove oil was 806 ± 29 mg/g (range, 776 to 844 mg/g) and that in cinnamon oil was 39.7 ± 2.0 mg/g (range, 37.9 to 42.2 mg/g). Addition-recovery tests were within the range of 86.9 to 98.8%.

Simultaneous Determination of the N‐Dealkylated Metabolites of Four Butyrophenone‐Type Agents in Rat Plasma by HPLC with Fluorescence Detection after Precolumn Derivatization with 4‐Fluoro‐7‐Nitro‐2,1,3‐Benzoxadiazole

Abstract The basic metabolites of butyrophenone type agents are sometimes more neurotoxic than the parent compounds. We have developed a high performance liquid chromatographic method with fluorescence detection (HPLC‐FL) to quantify the N‐dealkylated basic metabolites, i.e., 1,3‐dihydro‐1‐(1,2,3,6‐tetrahydro‐4‐pyridinyl) ‐2H‐benzimidazole‐2‐one, 1‐phenyl‐1,3,8‐triazaspiro[4.5]decan‐4‐one, 4‐(4‐chlorophenyl)‐4hydroxypiperidine, and 4‐(4‐bromophenyl)‐4‐hydroxypiperidine, of droperidol, spiperone, haloperidol, and bromperidol, respectively, in rat plasma after precolumn derivatization with 4‐fluoro‐7‐nitro‐2,1,3‐benzoxadiazole (NBD‐F). Mexiletine was carried through the procedure as an internal standard (IS). After liquid‐liquid extraction with benzene and evaporation, derivatization with NBD‐F was conducted in borate buffer (pH 8.5) at 60°C for 3 min. HPLC was conducted with a reversed‐phase (C18) column, eluted with a mixture of methanol‐water‐trifluoroacetic acid (600:400:0.4, v/v/v) at a flow rate of 1.0 mL/min at 25°C. The derivatives of four basic metabolites and the IS were well separated from each other in less than 48 min. The calibration curves were linear up to 0.5 µg/mL, and the lower limits of detection were 0.002 to 0.03 µg/mL. The coefficients of variation were less than 13.3%. These results confirm that HPLC‐FL after precolumn derivatization with NBD‐F is sensitive and satisfactory for the simultaneous assay of basic metabolites of four butyrophenone type agents in rat plasma.

Effect of imatinib mesilate on the disposition kinetics of ciclosporin in rats

The purpose of this study was to investigate the effect of imatinib mesilate on the disposition kinetics of ciclosporin in rats. The blood concentration‐time course and pharmacokinetic parameters of ciclosporin did not significantly change after intravenous injection of ciclosporin (10mg kg−1) in) rats treated with imatinib mesilate (50mg kg−1) as compared with a control. When ciclosporin (10mg kg−1) was orally administered, the time course, area under the curve, bioavailability and peak blood concentration of ciclosporin were significantly increased in rats that had been treated with imatinib mesilate 2 h before ciclosporin administration as compared with the control. Because both drugs are transported via P‐glycoprotein and breast cancer resistance protein and metabolized by cytochrome P450 3A2, the interaction of imatinib mesilate with these proteins may be responsible for the increased intestinal absorption of ciclosporin in rats. These results indicate that imatinib mesilate enhanced the intestinal absorption of ciclosporin in rats with only the oral administration of ciclosporin, suggesting that our results support clinical data. In addition, imatinib mesilate may increase the pharmacological effects and possibly toxicity of ciclosporin.

HPLC-UV Analysis of Phenol and Chlorophenols in Water After Precolumn Derivatization with 4-Fluoro-7-nitro-2,1,3-benzoxadiazole

Abstract Chlorophenols (2-chlorophenol, 4-chlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, and 2,4,6-trichlorophenol) are present in drinking water and tap water as a result of disinfection processes employing chlorination. In this study, the levels of phenol and the above chlorophenols in water (100 µL aliquots) were simultaneously analyzed by HPLC-UV (380 nm) after precolumn derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). Standard curves were obtained after derivatization with NBD-F in borate buffer (pH 8.5) at room temperature for 5 min. The six NBD-labeled compounds were well separated in less than 20 min. Plots were linear in the range of 0.01∼0.03 to 0.5 mg/L, with r 2 value ≥0.9953, for all compounds. The lower limits of detection were 0.004 to 0.01 mg/L (signal-to-noise ratio of 3:1). The coefficients of variation were less than 5.7%. The recovery values of phenol and chlorophenols from tap water and natural mineral water spiked with the standard mixture were satisfactory. While the levels of phenol and chlorophenols in water samples tested were below the lower limit of determination, our method is expected to be useful for identifying environmental water samples that are contaminated with phenol and chlorophenols, i.e., for testing whether or not official guidelines are met.

DETERMINATION OF FLUOXETINE ENANTIOMERS IN HUMAN SERUM AND URINE BY ISOCRATIC HPLC AND PRE-COLUMN FLUORESCENCE DERIVATIZATION WITH (R)-(+)-4-NITRO-7-(2-CHLOROFORMYLPYRROLIDIN-1-YL)-2,1,3-BENZOXADIAZOLE

Fluoxetine (FLX) is a highly selective serotonin reuptake inhibitor used as an antidepressant agent and administered as the racemate. In this study, we developed an isocratic HPLC method with fluorescence detection for the determination of FLX enantiomers ((R)-FLX and (S)-FLX) in human serum and urine, after pre-column derivatization with (R)-(+)-4-nitro-7-(2-chloroformyl- pyrrolidin-1-yl)-2,1,3-benzoxadiazole ((R)-(+)-NBD-Pro-COCl). After basic extraction of the samples into pentane, derivatization with (R)-(+)-NBD-Pro-COCl was conducted in borate buffer (pH 9.0) at 70°C for 5 min. Protriptyline was utilized as an internal standard. The regression equations for (R)-FLX hydrochloride and (S)-FLX hydrochloride in human serum showed good linearity in the range of 0.01–0.5 μg/mL with the detection limit of 0.005 μg/mL and in the range of 0.025–0.5 μg/mL with the detection limit of 0.008 μg/mL, respectively. The corresponding values for human urine were 0.025–0.5 μg/mL with the detection limit of 0.014 μg/mL and 0.025–0.5 μg/mL with the detection limit of 0.010 μg/mL, respectively. The coefficients of variation were less than 11.8%, and recovery was good. Our method using (R)-(+)-NBD-Pro-COCl is useful for simultaneous determination of (R)-FLX and (S)-FLX in human serum and urine, and may be suitable for therapeutic drug monitoring.

Simultaneous Determination of Four Basic Metabolites Formed from Butyrophenone Type Agents by HPLC with Dual Ultraviolet Detection

Abstract Some basic metabolites of butyrophenone‐type agents are more neurotoxic than the parent compounds. We have developed a high performance liquid chromatography method with dual ultraviolet detection (HPLC‐dual UV method) to quantify simultaneously the four basic metabolites {1,3‐dihydro‐1‐(1,2,3,6‐tetrahydro‐4‐pyridinyl)‐2H‐benzimidazole‐2‐one, (DTP) 1‐phenyl‐1,3,8‐triazaspiro[4.5] decan‐4‐one (PTS), 4‐(4‐chlorophenyl)‐4‐hydroxypiperidine (CPHP), and 4‐(4‐bromophenyl)‐4‐hydroxypiperidine (BPHP)} of droperidol, spiperone, haloperidol, and bromperidol, respectively. DTP, PTS, CPHP, and BPHP were monitored at their UV maxima (278, 247, 220, and 220 nm, respectively) and also at 200 nm. A reversed‐phase (C18) column was eluted with a mixture of acetonitrile‐water‐85% phosphoric acid (80:920:1, v/v/v) at a flow rate of 1.0 mL/min at 25°C. The four basic metabolites were well separated from each other in less than 25 min. The lower limits of detection were 8 to 20 ng/mL with detection at the UV absorption maxima, and 5 to 12 ng/mL with detection at 200 nm. The coefficients of variation for intra‐and inter‐day assays were less than 13.4%. The recoveries were satisfactory. These results confirm that the HPLC‐dual UV method is satisfactory for the simultaneous assay of DTP, PTS, CPHP, and BPHP in phosphate‐buffered saline.

Determination of three phenylphenols in grapefruit juice by HPLC after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole

Abstracto-Phenylphenol is generally utilized as a disinfectant for citrus fruits. The purpose of this study is to develop a high-performance liquid chromatography coupled with ultraviolet detection (380 nm) method for simultaneous determination of o-phenylphenol, and its analogues (m-phenylphenol and p-phenylphenol) in grapefruit juice after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). 2-Hydroxyfluorene was used as an internal standard (IS). Standard curves were obtained after derivatization with NBD-F in borate buffer (pH 8.0) at room temperature for 5 min. The three NBD-F derivatives were almost completely separated on a Cholester column (5 μm, 3.0 mm i.d. × 150 mm). Calibration plots were linear in the range of absolute amount of 1.04 ∼ 2.08 to 41.6 ng/50 μL injection volume, with r2 values ≥0.9981, for the three compounds. The lower limits of detection were 0.3 to 0.7 ng/50 μL injection volume (signal-to-noise ratio of 3 : 1). The coefficients of variation were less than 11.1%. After extraction of grapefruit juice (2.0 mL) with n-pentane, the level of o-phenylphenol in the juice was estimated to be 20.2 ± 2.0 ng/mL (n = 6, mean ± SD), while m-phenylphenol and p-phenylphenol were below the lower limits of quantification. The recovery values of the three phenylphenols from samples spiked with a standard mixture of authentic compounds and IS were satisfactory (99.1 to 118.7%).

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY COUPLED WITH FLUORESCENCE DETECTION FOR SIMULTANEOUS DETERMINATION OF HONOKIOL AND MAGNOLOL IN HANGE-KOBOKU-TO DRIED EXTRACT GRANULES

Honokiol and magnolol in Magnoliae Cortex, one of five medical herbal ingredients of Hange-koboku-to dried extract granules, have pharmacological activities, including decreasing anxiety and improving sleep. Here, we present a method employing aqueous extraction, followed by high-performance liquid chromatography with fluorescence detection, for the simultaneous determination of honokiol and magnolol in Hange-koboku-to dried extract granules. Excitation and emission wavelengths were set at 304 nm and 340 nm, respectively, for honokiol detection until 9 min, then the emission wavelength was shifted at 405 nm for magnolol detection. The regression equations for honokiol and magnolol in standard aqueous solutions showed good linearity in the range of 0.05–1 µg/mL with lower limits of detection (signal-to-noise ratio of 3) of 0.0047 and 0.0061 µg/mL, respectively. The coefficients of variation were less than 5.6%. It was found that Hange-koboku-to dried extract granules (1.00 g) contains honokiol (3.30 ± 0.21 mg; range, 3.08 to 3.63 mg) and magnolol (5.40 ± 0.37 mg; range, 4.80 to 5.80 mg) (mean ± S.D., n = 5). Recovery tests were satisfactory (90.7–104.8%).