荧光检测器(fluorescencedetector).ppt

1、HPLC-荧光检测器（HPLC-fluorescence detector）,1,应用,Liquid chromatographic assay with fluorescence detection to determine ajmaline in serum from patients with suspected Brugada syndrome Rolf W. Sparidansa，Journal of Chromatography B，2010,2,Contents,3,ajmaline,A diagnostic agent to reveal the lectrocardiogra

2、phic characteristics in patients with suspected Brugada syndrome.,The Brugada syndrome is an electrical heart disease that affects predominantly adult males (male to female ratio 8:1) and has a high prevalence in South-East Asia.,Introduction,4,To evaluate the relationship between the pharmacokineti

3、cs and the observed ECG characteristics after intravenous administration of ajmaline, an assay for this drug in human serum samples was required.,GCMS after an extremely tedious sample pre-treatment using five liquidliquid extraction steps and derivatization.,a simple bioanalytical assay for ajmalin

4、e using LCfluorescence detection,5,Chemicals,Ajmaline（fluka） Acetonitrile(gradient grade) Methanol (HPLC grade) Perchloric acid（analytical grade） Home-purified water Human serum（pooled and from individual donors）,6,equipment and conditions,Equipment ：Shimadzu LC system Column：a Sentry Guard Symmetry

5、C18 pre-column (3.9mm20mm, 5m ) 、a Waters Symmetry C18 column(4.6mm100mm, 3.5m)； 40 Mobile phase：pH 2.2perchloric acid-acetonitrile (77.5：22.5) flow rate 1.5 ml/min Detector ：a FP-920 fluorescence detector excitation wavelength 290nm;memission 355nm ;,7,100l Serum,mixied vigorously by vortex mixing,

6、50l 10% (w/v) perchloric acid,centrifuged for 10 min at 0 and 12103g,Supernatant into an insert in an injection vial,15l into the LC system,Sample pre-treatment,8,perchloric acid,low pH (ca. 2.2) protonates the residual silanol groups,ion pairing properties increase the retention of the basic analyt

7、e.,comparison between trichloroacetic Acid and acetonitrile,Perchloric acid was chosen as a simple precipitating agents,9,one stock solution,pooled human serum,5278 ng/ml stored at 80 ,2111, 528, 211, 52.8 and 21.1 ng/ml prepared daily,blank matrix,ajmaline,methanol,ca. 1.2 mg/ml Stock solutions sto

8、red at 4 ,Calibration,calibration curves ： the reversed square of the concentration of the analyte (1/x2) and the peak area of ajmaline.,10,Add Your Text,second stock solution,pooled human serum,QCsamples stored at 80 ,4160 (QC-high)、500 (QC-med)、50 (QC-low) 、25 (QC-LLQ) ng/ml,sextuple analysis of E

9、ach QC sample in three analytical runs on three separate days,Relative standard deviations within and between day precision,Precision and accuracy,11,Six individual serum samples,11 compounds,processed as blanks and after spiking with ajmaline at the LLQ level (25 ng/ml) and at 50 ng/ml,methadone, s

10、ulfamethoxazole, trimethoprim, ranitidine, caffeine, acetaminophen, oxazepam, rifampicin, pantoprazole, dexamethasone and metoclopramide,Blank serum samples,drug,near the upper therapeutic concentration,ajmaline at the LLQ level,Selectivity,12,Recovery,The extraction recovery was determined by compa

11、ring responses of processed samples (QC-high, -med and -low) with standard solutions at these levels in 25% (v/v) methanol,13,Stability,长期,冷冻-解冻循环3次,短期,Store 16h , 23 ,thawing at 23 during 12 h and freezing again at 80 for at least 1 day,8 months at 80 C,QC-high and low serum samples/Triplicate or q

12、uadruplicate analysis,溶液,storage 40 for 24 h and at 4 for 1 week,both stock solutions after 8 months storage at 4 ,QC-high, -med and -low serum samples,短期,处理后,14,Ten dosages to a second male subject (21 years of age, 90 kg body weight).,Nine aliquots of 2ml of 5 mg/ml to a male patient (77 years of

13、age, 90 kg body weight) with 1min intervals.,2,1,Patient samples,Blood samples were taken pre-dose and at approximately 1, 2, 5, 10, 15, 20, 30, 45, 60, 90, and 120 min after the last injection into glass tubes. After centrifugation at 3.5103g at ambient temperature for 5min, serum was separated and

14、 stored at 80 C until analysis with the presented assay,15,y = 1641(1263) + 758(34)x r= 0.9980.003 n=3 215300 ng/ml the lower limit of quantification was 25 ng/ml,16,Results and discussion,17,18,Selectivity,From the 11 drugs tested for potentially interference, sulfamethoxazole almost co- eluted wit

15、h ajmaline. All others did not interfere with the quantification of ajmaline.,extraction recoveries,QC-high57.70.1%, QC-med 56.11.4% QC-low 57.30.9% These relatively low recoveries showed excellent precisions and fulfilled the requirement of a bioanalytical assay,19,20,Pharmacokinetic curves of ajmaline in serum in two suspected Brugada syndrome patients during an ajmaline drug challenge test. (;) 21-year-old male; (; ) 77-year-old male.,21,A simple, accurate and precise bioanalytical assay for ajmaline has been pre