hplc determination of antidepressants and their ...validated method for analysis of enalapril in...
TRANSCRIPT
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Copyright 1998 Waters Corporation
HPLC Determination of Antidepressants and their
Metabolites in Plasma
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Copyright 1998 Waters Corporation
AbstractUsing 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 basesUsing the two-dimensional SPE method, we were able to selectively isolate analytes of interest from a complex sample matrix while eliminating almost all interferences.
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“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
Hydrophilicmonomer
Lipophilicmonomer
N O
Two Unique Properties Are Carefully Balanced
Reference: "A Novel 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
Oasis™ HLB SorbentHydro philic-Li pophilic Balanced
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Copyright 1998 Waters Corporation
Drying Effect on Recovery: C18 vs. Oasis™ HLB Cartridges
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C18 (1cc/100mg) HLB (1cc/30mg)
* No Impact of Sorbent Drying
* No Silanol Interaction
* No Breakthroughof Polar Analyte
Bouvier, Capparella
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Copyright 1998 Waters Corporation
One-Dimensional SPE Method
Simple, Rugged, and Highly Reproducible
One General SPE Method for a Wide Range of Compounds
Condition/Equilibrate1 mL methanol/1 mL water
Load1 mL spiked sample
Wash1 mL 5% methanol in water
Elute1 mL methanol
Evaporate and ReconstituteEvaporate and Reconstitute
Prepare Sample Solution
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Copyright 1998 Waters Corporation
O
CHCH2CH2N(CH3)H
O
CHCH2CH2N(CH3)2
CHCH2CH2NHCH3 CHCH2CH2N(CH3)2
AmitriptylineNortriptylineDoxepinNordoxepin
H2N
SNH
OO ON
CH3
H3CO
H3CO
OCH3
N
N
NH2
NH2
H2N
SN
OO
N
N
CH3
O
O
N COOHCH3
O
O
NCH3 O
O
O
O
NCH3 O
OCH3
N
NNH2
Cl N
HN
O
Cl
OHN
HN
Desmethyldiazepam(Nordiazepam)
O
Cl N
HN
O
Cl
Chlordiazepoxide
N
NNHCH3
Cl
Demoxepam
Benzoylecgonine Cocaine
Cocaine andMetabolites
Acid and Base
Cocaethylene
Oxazepam
OOO
CH3
Sulfamethoxazole Trimethoprim Sulfmerazine
Chlordiazepxideand Metabolites
Norchlordiazepoxide
Base
Cheng
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Copyright 1998 Waters Corporation
Bases: Tricyclic Antidepressants
Column: Symmetry® C18, 5 µm, 3.9 x 150 mm
Temperature: 35°CMobile Phase: 20 mM Phosphate pH 7:
Methanol (30:70)Detection: UV at 254 nmFlow Rate: 1.0 mL/min.Injection Volume: 20 µLSample Identification:
Peak 1: Nordoxepin (I.S.)Peak 2: NortriptylinePeak 3: DoxepinPeak 4: Amitriptyline
Compound Concentration % Recovery % RSD
Nortriptyline 0.5 µg/mL 99.7 2.3
Doxepin 0.5 µg/mL 94.0 1.3
Amitriptyline 1.0 µg/mL 102 2.5
5.00 10.0 15.0 20.0Minutes
1
2
3
4
0.00
6AU
Cheng
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Copyright 1998 Waters Corporation
Results from the One-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%
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Copyright 1998 Waters Corporation
Two-Dimensional SPE Method
Condition/Equilibrate1 mL methanol/1 mL water
Prepare Sample Solution
Load1 mL spiked sample solution
Wash1 mL 5% methanol in water
Washbase in methanol/water
Eluteacid in methanol/water
Washacid in methanol/water
Elutebase in methanol/water
Acids Bases
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Copyright 1998 Waters Corporation
Determination of % MeOH in 2-D SPE Method: Wash-Elute Study
Condition/Equilibrate 20 Cartridges/Wells1 mL methanol/1 mL water
Load1 mL spiked saline solution
Prepare Analytes in Saline
10 cartridges/wells 10 cartridges/wellsElute
acid in various % methanol in waterElute
base in various % methanol in water
e.g. 2% NH4OH e.g. 2% HAc
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Copyright 1998 Waters Corporation
Wash-Elute Study (Antidepressants):
0 20 40 60 80 100
% MeOH
0
50000
100000
150000
200000
Are
a (u
V *
sec
)
Wash (base)
0 20 40 60 80 100
% MeOH
0
50000
100000
150000
Are
a (u
V *
sec
) NordoxepinNortriptylineVerapamil, nor-MethylDoxepinAmitriptylineVerapamilVerapamil,Methoxy
Elute (acid)
Cheng
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Copyright 1998 Waters Corporation
Comparison of SPE Methods: Antidepressants in Plasma
heng
1
1
2
2
3
34
4 5
5
6
7
6
7
0.002 AU
0.002 AU
5 1510 Min.
5 10 Min.15
Sample
SampleBlank
One-Dimensional
Two-Dimensional
Blank
Column: Symmetry® RP8, 5 µm 3.9 x 150 mm
Temperature: 30°CMobile Phase: 100 mM Phosphate pH
acetonitrile:methanol (55:35:10)
Detection: UV at 230 nmFlow Rate: 1.0 mL/min.Injection Volume: 50 µL
Sample Identification:Peak 1: Nordoxepin Peak 2: NortriptylinePeak 3: NorverapamilPeak 4: DoxepinPeak 5: AmitriptylinePeak 6: VerapamilPeak 7: Methoxyverapamil (I.S.)
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Copyright 1998 Waters Corporation
Results: Antidepressants
Compound
Concentration [µg/mL]
Recovery(%)
RSD (%)(n=6)
Recovery(%)
RSD(n=3)
Amitriptyline 1.46 99.9 1.1 100.7 0.9
Doxepin 1.0 98.4 0.7 114 3.3
Nordoxepin 0.5 92.8 0.9 94.8 1.1
Nortriptyline 0.62 95.3 1.3 97.7 0.2
Verapamil 2.5 96.7 0.6 118 0.6
Norverapamil 1.0 95.9 0.6 97.3 1.0
Cheng
2-Dimensional 1-Dimensional
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Oasis™HLB Extraction Plates and Cartridges
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Copyright 1998 Waters Corporation
Prepare Sample1 mL spiked porcine serum
Condition and equilibrate1 mL methanol and 1 mL water
Load SampleWash
1 mL 40% methanol containing 2% ammonium hydroxide
Elute 500 µL 80% methanol containing 2% acetic acid
Add 500 µL 2% ammonium hydroxide
Oasis™ 96-Well Extraction Plate for High Throughput Analysis: Trazodone and Metabolite
HN N
Cl
1-(3-Chlorophenyl)piperazine dihydrochloride
NN
N
O
N N
Cl
Trazodone
Cheng
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Copyright 1998 Waters Corporation
Results from Oasis™ 96-Well Extraction Plate and Oasis™
Extraction CartridgesRecoveries all greater than 88%Reproducibility all less than 5.6%
Column: Symmetry® RP8, 5 µm, 3.9 x 150 mmTemperature: 30°CMobile Phase: 50 mM Phosphate pH 7: :methanol (36:64)Detection: UV at 250 nmFlow Rate: 1.0 mL/min.
Injection Volume: 25 µLPeak 1: 1-(3-chlorophenyl)peperazinePeak 2: Maprotiline (I.S.)Peak 3: Trazodone
0.01 AU
1 654320minute
1
2
3
blank
standard
sample
Cheng
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Copyright 1998 Waters Corporation
single moldsmaller lower stage for low bed masseslarger upper stage for high bed massescurrent bed configuration maintained
30 mg10 mg 60 mg
Low Elution VolumeWaters Proprietary* Two-Stage Well Design
* patent pending
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Copyright 1998 Waters Corporation
Low Elut ion Volume Test Mixture: Acids , Neutrals, and Bases
(Arranged from Most to Least Polar)
Betamethasone ValerateO
HO
F
OCO(CH2)3CH3
OH
O
Betamethasone
O
HO
F
OHOH
O
HO
HN
O
Acetaminophen Procainamide
OS
HN
HN
N
CHNO2 H2N
NH
N
O
CH3
CH3
Ranitidine
OHN
OH
Propranolol
O
CHCH2CH2N(CH3)2
Doxepin
O
O
O
O
Dipropyl Phthalate
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Copyright 1998 Waters Corporation
0 5 10 15
ProcainamideRanitidineAcetaminophenTheophyllineBetamethasonePropranololDipropyl phth.DoxepinB-meth Val
n = 8 wells
Waters 10 mg Extraction Plate:% RSDs in 150 µL Elution Volume
Waters 10 mg Plate
3MC18-SD
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Cop
yrig
ht 1
998
Wat
ers
Cor
pora
tion
test
Enalapril
Enalapril -d5(IS)
Enalaprilat
Enalaprilat -d5(IS)
PPD Pharmaco
High Sensitivity DectectionEnalapril and Enalaprilat in Human Plasma (BLANKS)
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Cop
yrig
ht 1
998
Wat
ers
Cor
pora
tion
test
0.25 ng/mLEnalapril
5 ng/mLEnalapril -d5(IS)
0.25 ng/mLEnalaprilat
5 ng/mLEnalaprilat -d5(IS)
PPD Pharmaco
High Sensitivity DectectionEnalapril and Enalaprilat in Human Plasma
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Copyright 1998 Waters Corporation
Validated Method for Analysis of Enalapril in Human Plasma
Concentration % Recoveryn=6
Interday Precision, %CV
n= 5 (x 2)
Intraday Precision%CV n=6
0.5 ng/mL 110.9 5.2 3.65 ng/mL 112.2 2.2 4.380 ng/mL 107.0 1.6 1.6
Internal Std. 91.1
PPD Pharmaco
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Copyright 1998 Waters Corporation
Validated Method for Analysis of Enalaprilat in Human Plasma
Concentration % Recoveryn=6
Interday Precision, %CV
n= 5 (x 2)
Intraday Precision%CV n=6
0.5 ng/mL 103.6 5.2 3.05 ng/mL 109.4 3.4 3.280 ng/mL 104.5 1.7 1.2
Internal Std. 94.3
PPD Pharmaco
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Copyright 1998 Waters Corporation
SummaryUnique Sorbent
No impact of dryingNo silanolsExtended pH rangeUniversal sorbent
Fast and Easy Method Development
One-dimensional methodSimple methodOne method, One result
Two-dimensional methodStraightforward methodCleaner extracts