A rapid, selective and sensitive reverse-phase HPLC methods for the determination of atenolol and chlorthalidone in human serum and whole blood were validated, and applied to the pharmacokinetic study of atenolol and chlorthalidone combination therapy. Atenolol and an internal standard, pindolol, we...
A rapid, selective and sensitive reverse-phase HPLC methods for the determination of atenolol and chlorthalidone in human serum and whole blood were validated, and applied to the pharmacokinetic study of atenolol and chlorthalidone combination therapy. Atenolol and an internal standard, pindolol, were extracted from human serum by liquid-liquid extraction, and analyzed on a $\mu$-Bondapak C18 $10-{\mu}$ column in a mobile phase of methanol-0.01 M potassium dihydrogenphosphate(30:70, v/v, adjusted to pH 3.5) and fluorescence detection(emission: 300 nm, excitation: 224 nm). Chlorthalidone and an internal standard, probenecid, were extracted form human whole blood by liquid-liquid extraction, and analyzed on a Luna C18 $5-{\mu}$ column in a mobile phase of acetonitrile containing 77% 0.01 M sodium acetate and UV detection at 214 nm. These analysis were performed at three different laboratories using the same quality control(QC) samples. The chromatograms showed good resolution, sensitivity, and no interference by human serum and whole blood, respectively. The methods showed linear responses over a concentration range of 10-1,000 ng/mL for atenolol and 0.05-20 ${\mu}g/mL$ for chlorthalidone, with correlation coefficients of greater than 0.999 at all the three laboratories. Intra- and inter-day assay precision and accuracy fulfilled international requirements. Stability studies(freeze-thaw, short-, long-term, extracted sample and stock solution) showed that atenolol and chlorthalidone were stable. The lower limit of quantitation of atenolol and chlorthalidone were 10 ng/mL and 0.05 ${\mu}g/mL$, respectively, which was sensitive enough for pharmacokinetic studies. These methods were applied to the pharmacokinetic study of atenolol and chlorthalidone in human volunteers following a single oral administration of Hyundai $Tenoretic^{\circledR}$ tablet(atenolol 50 mg and chlorthalidone 12.5 mg) at three different laboratories.
A rapid, selective and sensitive reverse-phase HPLC methods for the determination of atenolol and chlorthalidone in human serum and whole blood were validated, and applied to the pharmacokinetic study of atenolol and chlorthalidone combination therapy. Atenolol and an internal standard, pindolol, were extracted from human serum by liquid-liquid extraction, and analyzed on a $\mu$-Bondapak C18 $10-{\mu}$ column in a mobile phase of methanol-0.01 M potassium dihydrogenphosphate(30:70, v/v, adjusted to pH 3.5) and fluorescence detection(emission: 300 nm, excitation: 224 nm). Chlorthalidone and an internal standard, probenecid, were extracted form human whole blood by liquid-liquid extraction, and analyzed on a Luna C18 $5-{\mu}$ column in a mobile phase of acetonitrile containing 77% 0.01 M sodium acetate and UV detection at 214 nm. These analysis were performed at three different laboratories using the same quality control(QC) samples. The chromatograms showed good resolution, sensitivity, and no interference by human serum and whole blood, respectively. The methods showed linear responses over a concentration range of 10-1,000 ng/mL for atenolol and 0.05-20 ${\mu}g/mL$ for chlorthalidone, with correlation coefficients of greater than 0.999 at all the three laboratories. Intra- and inter-day assay precision and accuracy fulfilled international requirements. Stability studies(freeze-thaw, short-, long-term, extracted sample and stock solution) showed that atenolol and chlorthalidone were stable. The lower limit of quantitation of atenolol and chlorthalidone were 10 ng/mL and 0.05 ${\mu}g/mL$, respectively, which was sensitive enough for pharmacokinetic studies. These methods were applied to the pharmacokinetic study of atenolol and chlorthalidone in human volunteers following a single oral administration of Hyundai $Tenoretic^{\circledR}$ tablet(atenolol 50 mg and chlorthalidone 12.5 mg) at three different laboratories.
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제안 방법
0 cm). All subjects were selected after passing a clinical screening procedure including a physical examination and lab oratory tests (blood analysis; hemoglobin, hematocrit, RBC, WBC, platelet, differential counting of WBC, total proteins, albumin, sGOT, sGPT, alkaline phosphatase, total bilirubin, cholesterol, creatinine, blood urea nitrogen, and glucose fasting and urine analysis; specific gravity, color, pH, sugar, albumin, bilirubin, RBC, WBC and cast). These measures were nec essary to ensure that the existing degree of variation would not be due to an influence of disease or other medications.
was identical for all the subjects. Blood samples were collected in two vacutainer tubes before dosing and at 0.5, 1, 2, 3, 4, 6, 8, 12, 24, 48, 72, 96 and 120 h after drug admin istration. Following centrifugation (3, 000 rpm, 20 min, 4℃), serum samples for atenolol analysis were transferred to poly ethylene tubes and immediately stored at -80℃ until analysis.
Serum calibration standards of atenolol were prepared at concentrations of 10, 20, 50, 100, 200, 500 and 1000 ng/mL in drug-free pooled serum obtained from twelve different volunteers. In the same manner, quality control (QC) serum samples at low (20 ng/mL), medium (100 ng/mL), and high (500 ng/mL), were prepared to evaluate accuracy and precision. All solutions and samples of atenolol and pindolol were protected against light during manipulations.
5, 2, 5, 10 and 20 μg/mL in drug-free pooled whole blood obtained from twelve different volunteers. In the same manner, quality control (QC) whole blood samples at low (0.1 μg/mL), medium (0.5 μg/mL), and high (5 μg/mL), were prepared to evaluate accuracy and precision. All solutions and samples of chlorthalidone and probenecid were protected against light during manipulations.
prepared as described above. The intra-day precision of the assay was assessed by calculating the coefficients of vari ation (CV) for the analysis of QC samples in five replicates, and inter-day precision was determined by the analysis of QC samples on five consecutive days. Accuacy was determined by comparing the calculated and known concentrations.
대상 데이터
For atenolol analysis, it consisted of two pumps (model LC-10AD), a degasser (model DGU-12A), and a fluorescence detector (model RF-10Axl, emission: 300 nm, excitation: 224 nm). The separation was performed on a g-Bondapak C18 col umn (10-μm particle size, 3.
이론/모형
14) The study protocol was approved by each Institutional Review Board (IRB). This study was performed according to the revised Declaration of Helsinki for biomedical research involving human subjects and the rules of Good Clinical Practice.
성능/효과
Accuacy was determined by comparing the calculated and known concentrations. The evaluation of precision was based on the criteria that relative standard deviation for each concentration level should not be more than ±15% except for the LLOQ, for which it should not exceed ±20%. Similarly, for accuracy, the mean value should not deviate by ±15% of the nominal concentration except for the LLOQ, where the limit was ±20%.
The mean percent accuracy value for serum samples was 105.99% and precision coefficient of variation (CV) was below 10.62% at the LLOQ (Table Ⅰ). The LLOQ value for serum was lower than that reported by Wolf-Coporda et al.
05 |ig/mL for chlorthalidone injected on-column with a 20 μL loop. The mean percent accuracy value for whole blood samples was 117.43% and precision coefficient of variation (CV) was below 14.63% at the LLOQ (Table Ⅱ). The LLOQ value for whole blood was lower than that reported by Rosen berg et al4) Furthermore, that method used a relatively large injection volume.
참고문헌 (16)
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