HPLC Determination of Antidepressants and their Metabolites in Plasma

Transcription

1 HPLC Determination of Antidepressants and their Metabolites in Plasma <XQJ)RQJ&KHQJ, Laura Bean, Edouard S. P. Bouvier, Mark Capparella, Pamela Iraneta, Andy Jarrell, Uwe D. eue, Dorothy J. Phillips, and Doug Wittmer Waters Corporation, 34 Maple Street, Milford, MA 01757

2 Abstract Using the one-dimensional SPE method, we were able to obtain very high recoveries (all greater than 86%) and excellent reproducibility (all less than 5.0%) for a wide range of drugs. These compounds included acids, neutrals, and bases Using the two-dimensional SPE method, we were able to selectively isolate analytes of interest from a complex sample matrix while eliminating almost all interferences.

3 asis HLB Sorbent Hydrophilic-Lipophilic Balanced Hydrophilic monomer Lipophilic monomer water loving Provides the correct wetting property to keep the surface of the packing solvated even when the cartridges run dry fat loving Provides reversed-phase property for analyte retention Two Unique Properties Are Carefully Balanced Reference: "A ovel Polymeric Reversed-Phase Sorbent for Solid-Phase Extraction," LC GC Int. 10 (9) E.S.P. Bouvier, D.M. Martin, P.C. Iraneta, M. Capparella, Y. Cheng, and D.J. Phillips

4 3HUFHQW UHFRYHU\ Drying Effect on Recovery: C 18 C 18 (1cc/100mg) vs. asis HLB Cartridges 'U\LQJ 7LPH PLQXWHV HLB (1cc/30mg) 'U\LQJ 7LPH PLQXWHV * o Impact of Sorbent Drying * o Silanol Interaction * o Breakthrough of Polar Analyte Bouvier, Capparella 3URFDLQDPLGH $FHWDPLQRSKHQ 5DQLWLGLQH 3URSUDQRR 'R[HSLQ

5 ne-dimensional SPE Method Prepare Sample Solution Simple, Rugged, and Highly Reproducible ne General SPE Method for a Wide Range of Compounds Condition/Equilibrate 1 ml methanol/1 ml water Load 1 ml spiked sample Wash 1 ml 5% methanol in water Elute 1 ml methanol Evaporate and Reconstitute

6 Base CHCH 2 CH 2 (CH 3 )H CHCH 2 CH 2 (CH 3 ) 2 ordoxepin Doxepin ortriptyline CHCH 2 CH 2 HCH 3 CHCH 2 CH 2 (CH 3 ) 2 Amitriptyline Acid and Base H 2 S H CH 3 H 3 C H 3 C CH 3 H 2 H 2 H 2 S CH 3 Sulfamethoxazole Trimethoprim Sulfmerazine Chlordiazepxide and Metabolites Cl H 2 Cl H H Cl H Cl HCH 3 Cl H orchlordiazepoxide xazepam Desmethyldiazepam (ordiazepam) Chlordiazepoxide Demoxepam Cocaine and Metabolites CH 3 CH CH 3 CH 3 CH 3 CH 3 Cheng Benzoylecgonine Cocaine Cocaethylene

7 Bases: Tricyclic Antidepressants Compound Concentration % Recovery % RSD ortriptyline 0.5 µg/ml Doxepin 0.5 µg/ml Amitriptyline 1.0 µg/ml AU Column: Symmetry C18, 5 µm, 3.9 x 150 mm Temperature: 35 C Mobile Phase: 20 mm Phosphate ph 7: Methanol (30:70) Detection: UV at 254 nm Flow Rate: 1.0 ml/min. Injection Volume: 20 µl Sample Identification: Peak 1: ordoxepin (I.S.) Peak 2: ortriptyline Peak 3: Doxepin Peak 4: Amitriptyline Cheng Minutes

8 Results from the ne-dimensional SPE Method Using the one-dimensional SPE method, we were able to obtain excellent results for a wide range of compounds (concentration ranging between 40 ng/ml and 4.0 µg/ml) from a serum matrix: Recoveries all greater than 86% Reproducibility all less than 5.0%

9 Two-Dimensional SPE Method Prepare Sample Solution Condition/Equilibrate 1 ml methanol/1 ml water Load 1 ml spiked sample solution Acids Wash 1 ml 5% methanol in water Bases Wash acid in methanol/water Wash base in methanol/water Elute base in methanol/water Elute acid in methanol/water

10 Determination of % MeH in 2-D SPE Method: Wash-Elute Study Prepare Analytes in Saline Condition/Equilibrate 20 Cartridges/Wells 1 ml methanol/1 ml water Load 1 ml spiked saline solution 10 cartridges/wells 10 cartridges/wells Elute base in various % methanol in water e.g. 2% H4H Elute acid in various % methanol in water e.g. 2% HAc

11 Wash-Elute Study (Antidepressants): Area (uv * sec) Wash (base) Area (uv * sec) Cheng % MeH Elute (acid) % MeH ordoxepin ortriptyline Verapamil, nor- Methyl Doxepin Amitriptyline Verapamil Verapamil, Methoxy

12 Comparison of SPE Methods: heng Antidepressants in Plasma AU Min AU Min. 6 ne-dimensional 7 7 Sample Blank Two-Dimensional Sample Blank Column: Symmetry RP8, 5 µm 3.9 x 150 mm Temperature: 30 C Mobile Phase: 100 mm Phosphate ph acetonitrile:methanol (55:35:10) Detection: UV at 230 nm Flow Rate: 1.0 ml/min. Injection Volume: 50 µl Sample Identification: Peak 1: ordoxepin Peak 2: ortriptyline Peak 3: orverapamil Peak 4: Doxepin Peak 5: Amitriptyline Peak 6: Verapamil Peak 7: Methoxyverapamil (I.S.)

13 Results: Antidepressants Compound 2-Dimensional 1-Dimensional Concentration [µg/ml] Recovery (%) RSD (%) (n=6) Recovery (%) RSD (n=3) Amitriptyline Doxepin ordoxepin ortriptyline Verapamil orverapamil Cheng

14 asis HLB Extraction Plates and Cartridges

15 asis 96-Well Extraction Plate for High Throughput Analysis: Trazodone and Metabolite Cheng H 1-(3-Chlorophenyl)piperazine dihydrochloride Trazodone Cl Cl Prepare Sample 1 ml spiked porcine serum Condition and equilibrate 1 ml methanol and 1 ml water Load Sample Wash 1 ml 40% methanol containing 2% ammonium hydroxide Elute 500 µl 80% methanol containing 2% acetic acid Add 500 µl 2% ammonium hydroxide

16 Results from asis 96-Well Extraction Plate and asis Extraction Cartridges 0.01 AU standard 0 sample blank Cheng minute 2 3 Recoveries all greater than 88% Reproducibility all less than 5.6% Column: Symmetry RP8, 5 µm, 3.9 x 150 mm Temperature: 30 C Mobile Phase: 50 mm Phosphate ph 7: :methanol (36:64) Detection: Flow Rate: UV at 250 nm 1.0 ml/min. Injection Volume: 25 µl Peak 1: 1-(3-chlorophenyl)peperazine Peak 2: Maprotiline (I.S.) Peak 3: Trazodone

17 Low Elution Volume Waters Proprietary* Two-Stage Well Design single mold smaller lower stage for low bed masses larger upper stage for high bed masses current bed configuration maintained 10 mg 30 mg 60 mg * patent pending

18 Low Elution Volume Test Mixture: Acids, eutrals, and Bases (Arranged from Most to Least Polar) H H Acetaminophen S Ranitidine H H CH 2 H 2 H Procainamide CH 3 CH 3 H H H H H F Betamethasone Propranolol Dipropyl Phthalate CHCH 2 CH 2 (CH 3 ) 2 H H C(CH 2 ) 3 CH 3 F Doxepin Betamethasone Valerate

19 Waters 10 mg Extraction Plate: % RSDs in 150 µl Elution Volume n = 8 wells 3M C18-SD Waters 10 mg Plate Procainamide Ranitidine Acetaminophen Theophylline Betamethasone Propranolol Dipropyl phth. Doxepin B-meth Val

20 High Sensitivity Dectection Enalapril and Enalaprilat in Human Plasma (BLAKS) Enalapril Enalapril -d5 (IS) Enalaprilat test Enalaprilat -d5 (IS) PPD Pharmaco

21 High Sensitivity Dectection Enalapril and Enalaprilat in Human Plasma 0.25 ng/ml Enalapril test 5 ng/ml Enalapril -d5 (IS) 0.25 ng/ml Enalaprilat 5 ng/ml Enalaprilat -d5 (IS) PPD Pharmaco

22 Validated Method for Analysis of Enalapril in Human Plasma Concentration % Recovery n=6 Interday Precision, %CV n= 5 (x 2) Intraday Precision %CV n=6 0.5 ng/ml ng/ml ng/ml Internal Std PPD Pharmaco

23 Validated Method for Analysis of Enalaprilat in Human Plasma Concentration % Recovery n=6 Interday Precision, %CV n= 5 (x 2) Intraday Precision %CV n=6 0.5 ng/ml ng/ml ng/ml Internal Std PPD Pharmaco

24 Summary Unique Sorbent o impact of drying o silanols Extended ph range Universal sorbent Fast and Easy Method Development ne-dimensional method Simple method ne method, ne result Two-dimensional method Straightforward method Cleaner extracts