Study On Pharmaceutical Analytic Methods In Human Aqueous Humor & Milk

It provides useful suggestions and evaluations about drug quality through clinical processes with the information acquired by using modern analytic methods.This present thesis was composed of two parts.PartⅠ-By measuring drug concentration in breast milk,provides data foundation for the rational drug use in lactation.PartⅡ-By measuring drug concentration of different dosage forms in aqueous humor,the paper preliminarily discusses the efficacy of different dosage forms.PartⅠ:A reliable and sensitive method for the determination of gatifloxacin in human aqueous humor was developed and validated.A 50μL aliquot of whole aqueous humor was extracted using liquid-liquid extraction after addition of ciprofloxacin as an internal standard.Analysis was performed on a Agilent 6410 QQQ LC-MS/MS in single ion monitoring positive mode using Electrospray Ionization(ESI) as an interface.The extracted ion for gatifloxacin was m/z 376.1→332.1 and for ciprofloxacin was m/z 332.1→288.1.Separation was achieved on a A ZORBAX Eclipse Plus C18 column (2.1×100 mm,1.8μm) using a mobile phase composed of 0.1mol·L^-1 formic acid aqueous-acetonitrile with 0.1mol·L^-1 formic acid at a flow rate of 0.3mL·min^-1.The mobile phase was delivered in gradient mode.The retention times of gatifloxacin and ciprofloxacin were 7.1 and 3.5 min,respectively.A good linear relationship was obtained between the peak area and two calibration curves were acquired.One is over the range of 0.5 to 12ng·mL^-1,the calibration curve equation is Y=1.9291X+0.6016(r=0.9964).The analysis of quality control samples for gatifloxacin at 1.0,6.0 and 40.0 ng/mL demonstrated excellent precision with relative standard deviation of 20.3%,9.1%and 9.5%respectively(n=5).Recoveries at concentrations of 1.0,6.0 and 40 ng/mL was 114.1%,106.5%,83.2%respectively.The other is over the range of 10.0 to 200.0ng·mL^-1, the calibration curve equation is Y=1.2665X+3.5886(r=0.9983).The analysis of quality control samples for gatifloxacin at 1.0,12.0 and 150.0 ng/mL demonstrated excellent precision with relative standard deviation of 20.3%,8.4%and 5.6%respectively(n=5). Recoveries at concentrations of 1.0,12.0 and 150 ng/mL was 114.1%,101.1%,102.0% respectively.No interference peaks or matrix effects were observed.This LC-MS/MS method for the determination of gatifioxacin in human aqueous humor has excellent specifications for sensitivity,reproducibility and accuracy.This LC-MS/MS technique was found to improve the quantitation of gatifloxacin in whole aqueous humor allowing its use in pharmacokinetic studies with clinically relevant doses.The built analysis methods were applied in 34 patients using eye drops and 40 patients using gel.After two dosage forms were applied,we calculated and compared of drug concentration in aqueous humor.The results showed individual difference clearly, besides,it is clear that gatifloxacin gel has a higher drug concentration in aqueous humor while comparing with gatifloxac eye drops,and the drug concentration of gatifloxacin gel maintains high level in aqueous humor and prolongs action time.Thus,Gatifloxacin gel has a good clinical effect.PartⅡ:A specific and accurate reversed-phase high performance liquid chromatography with UV detection was developed for the assay of Cefuroxime Sodium in secreted milk following injection administration of CS in mastitis patients.Cefuroxime was extracted by protein precipitation,the extracts were injected into a Agilent ZORBAX SB-C18 column(4.6mm×150mm,5μm) with UV detector set at 254nm wavelength.The mobile phase consisted of sodium acetate buffer(pH3.7) -acetonitrile(7:1) at a flow rate of 1.0 mL·min^-1,the column was at 30℃.The results show that the blank milk did not interfere with the determination of Cefuroxime Sodium.A good linear relationship was obtained between the peak area and the concentration of Cefuroxime Sodium over the range of 0.03 to 10μg·mL^-1.The linear equation was Y=16872X-273.38(r=0.9980).The limit of determination was 0.03μg·mL^-1.The average of recoveries was 110.2%,93.2%, 102.0%.The RSD was 4.6%,3.2%9.1%.This HPLC method for the determination of cefuroxime sodium in human milk has excellent specifications for sensitivity, reproducibility and accuracy.A specific and accurate reversed-phase high performance liquid chromatography with UV detection was developed for the assay of Levofloxacin in secreted milk following injection administration of Levofloxacin in mastitis patients.Levofloxacin was extracted by protein precipitation,the extracts were injected into a Agilent ZORBAX SB-C18 column(4.6mm×150mm,5μm) with UV detector set at 293nm wavelength.The mobile phase consisted of 0.05mol·L^-1 citric acid aqueous(pH3.4 corrected by Triethylamine) -acetonitrile(85:15)at a flow rate of 1.0 mL·min^-1,the column was at 30℃.The results show that the blank milk did not interfere with the determination of Levofloxacin.A good linear relationship was obtained between the peak area and the concentration of Levofloxacin over the range of 0.03 to 15.5μg·mL^-1.The linear equation was Y=48472X+4155.2(r=0.9988).The limit of determination was 0.02μg·mL^-1.The average of recoveries was 108.6%,107.2%,109.1%.The RSD was 4.5%,4.7%,5.5%. This HPLC method for the determination of levofloxacin in human milk has excellent specifications for sensitivity,reproducibility and accuracy.At present,cefuroxime sodium and levofloxacin are common drugs for curing mastitis cases.With establishment of analytic methods for drug in breast milk,it is helpful to understand the diminishing situation of the drug in breast milk and improve security of breast feeding.