Determination of hydrochlorothiazide concentration and pharmacokinetics in human plasma by liquid chromatography
Abstract: Hydrochlorothiazide is a diuretic and antihypertensive drug. Mainly suitable for cardiogenic edema, hepatogenic edema and renal edema: such as nephrotic syndrome, acute glomerulonephritis, chronic renal failure, and edema caused by excessive adrenal corticosteroids and estrogen; hypertension; diabetes insipidus disease.
1 Instruments
Shimadzu high performance liquid chromatograph: LC-10Advp pump, SP-10Avp ultraviolet detector, CTO-10Asvp column thermostat, VPclass5.0 chromatographic data workstation HCTZ tablets; HCTZ chemical reference; caffeine (internal standard; acetonitrile, methyl Tert-butyl ether is chromatographically pure; sodium octanesulfonate triethylamine and glacial acetic acid are analytically pure; water is water for injection.
2.1 Chromatographic separation conditions Chromatographic column: Diamond (200film × 4.6mm, 5m); mobile phase: acetonitrile-O. 15% sodium octanesulfonate (17:83), containing 1% glacial acetic acid and 0.05% triethylamine; flow rate: 1.0 mL · min. . ; Detection wavelength: 272nm; Column temperature: 40 ℃.
2.2 Subjects and trial design 10 male healthy volunteers, age (22.3 ± 0.6) years old, weight (65.4 ± 2.7) kg, normal liver and kidney function and ECG, before the test During the week and the trial period, any other drugs were banned. During the trial period, smoking, alcohol, and tea were banned, and an informed consent was signed. The trial was approved by the hospital medical ethics committee. After fasting for 12h, take HCTZ tablets 25mg on the next morning Et fasting El, before and after administration 0.25, 0.5, 0.75, 1.0, 1.5, 2.0,
4. 0, 8. 0, 12. 0, 24. 0, 36.0 h 5 mL of blood was taken in a vein, placed in a heparin anticoagulation tube, centrifuged at 3500 r · min for 10 min, and the separated plasma was stored in a refrigerator at -20 ° C.
2.3 Pretreatment of plasma samples Take 1.0 mL of plasma in a 10 mL glass centrifuge tube with a stopper, add 4.0 mg · L internal standard 50txL, 5 mL of methyl tert-butyl ether, vortex for 2 min, and centrifuge at 3000 r · min At 10 min, 4 mL of the methyl tert-butyl ether layer was taken, and nitrogen gas was swung under a water bath at 50 ° C., 200 txL of mobile phase was added to dissolve, and the supernatant 50 was injected for quantitative analysis of peak area.
2.4 In vivo analysis methodological evaluation The healthy blank plasma was precisely prepared into 3.8, 9.5, 38.0, 95.0, 190.0, 380. 0mg · L concentration of HCTZ standard plasma samples, according to 2 .3 method operation to prepare plasma standard curve. In addition, prepare 4.0, 40.0, 200, 0txg · L three kinds of HCTZ plasma quality control samples with different concentrations, operate according to the method of 2.3, and investigate the recovery rate and precision between Et and Et.
3 Discussion
In this study, a high-performance liquid chromatography method for determining the concentration of hydrochlorothiazide in plasma was established. The C column was used to separate the ion-pair chromatography. The peak shape had good symmetry, fast peaks, and high sensitivity. Using methyl tert-butyl ether extraction, the extraction is complete, with less interference from impurities. Experiments show that this method is simple and fast, and the recovery rate and the precision of intra-day and inter-day can be used for the pharmacokinetic study of the drug. After taking a single dose of hydrochlorothiazide in 10 healthy volunteers, it was absorbed quickly and started to work quickly.
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