BA-BE-BMF0120

Prévia do material em texto

1
Marcelo N. Muscará
muscara@usp.br
BMF0120 – Farmacologia geral
“Biodisponibilidade e
Bioequivalência”
1
Vias de administração
è Intra vascular
üIntra-venosa
üIntra-arterial
èExtra vascular
ü Sub-lingual
ü Oral
ü Sub-cutânea
ü Intramuscular
ü Intraperitoneal
ü Retal
ü Tópica
ü Inalatória
2
2
Fatores que afetam a absorção gastrintestinal
ü Motilidade gastrintestinal;
ü fluxo sangüíneo esplâncnico;
ü fatores físico-químicos (interações entre substãncias)
(ex: tetraciclina liga Ca2+Þ alimentos ricos em Ca2+ impedem 
sua absorção);
ü tamanho das partículas e tipo de formulação.
3
Biodisponibilidade
Extensão e velocidade de absorção do fármaco a 
partir de uma forma farmacêutica, derivados a 
partir da curva de concentração (circulação 
sistémica) em função do tempo.
F =
ASCe.v. x Dosei.v.
ASCi.v. x Dosee.v.
(0 < F < 1)
ASC: área sob a curva de concentração sanguínea do fármaco vs. tempo.
4
3
5
Eur J Clin Pharmacol (1996) 50 : 399–405 © Springer-Verlag 1996
P H A R M AC O K I N E T I C S A N D D IS P O S I T I O N
W. Ribeiro · M. N. Muscará · A. R. Martins 
H. Moreno Jr · G. B. Mendes · G. de Nucci
Bioequivalence s tudy of two enalapr i l maleate tablet formulat ions
in healthy male volunteers
Pharmacok inet ic versus pharmacodynamic approach
Received: 29 May 1995 / Accepted in revised form: 30 October 1995
Abstract Objective: Two different conventional release
enalapril maleate tablet formulations were evaluated
for their relative bioavailability (Eupressin tablets
10 mg, Biosintética as the test formulation vs Renitec
tablets 10 mg Merck Sharp & Dhome, as the reference
formulation). A single 20 mg oral dose of each prepa-
ration was administered to 18 healthy male adult vol-
unteers and their bioequivalence was assessed by
comparing the serum enalaprilat and total enalapril
(enalaprilat plus enalapril maleate) concentration-time
curves. Angiotensin converting enzyme (ACE) activity
was also quantified in each serum sample.
Results: The pharmacokinetic parameters obtained for
each formulation were the area under the time-
concentration curve from 0 to 24 h (AUC[0–24]), max-
imum concentration Cmax and the time at which it
occurred (tmax). When serum enalaprilat concentration-
time curves were employed to assess bioequivalence,
the formulations were bioequivalent in the extent but
not in the rate of absorption. However, no difference
in either the extent or the rate of absorption were
observed when serum total enalapril vs time curves
were analysed. ACE activity-time curves were similar
for both formulations and showed that ACE was 90%
inhibited 3–5 h after enalapril administration, and till
approximately 50% after 24 h. At that time, circulat-
ing enalaprilat and total enalapril levels were less than
the tenth of Cmax. 
Conclusion: The results show that complete bioequiv-
alence of the two formulations can be concluded from
serum total enalapril concentration data, and that
serum ACE activity is not a suitable pharmacodynamic
variable for assessing bioequivalence.
Key words Bioavailability, Enalapril formulations;
pharmacokinetics, ACE-activity, human
Introduct ion
Enalaprilat (MK422, N-[(S)-1-carboxy-3-phenyl-
propyl]-L-alanyl-L-proline) is a potent angiotensin
converting enzyme (ACE) inhibitor, widely used in
essential and renovascular hypertension [1, 2]. It
reduces the plasma levels of angiotensin II and aldo-
sterone [3, 4, 5] and increases endogenous bradykinin
concentrations [6, 7].
Enalaprilat is sold as the maleate ester (MK421,
enalapril), which has enhanced gastrointestinal absorp-
tion [2]. About 60% of a dose of enalapril is absorbed
after oral administration. The compound itself is not
a potent ACE inhibitor but its subsequent in vivo
hydrolysis in the liver results in production of the active
diacid form, enalaprilat, which itself is poorly absorbed
[1, 8]. The peak plasma concentration of the pro-drug
is generally observed about 1 h after oral administra-
tion, whereas the Cmax of enalaprilat occurs after 2–4 h
[2, 9, 10].
A significant proportion of enalaprilat (43%) is elim-
inated in the urine [11, 12, 13]. Elimination is biphasic,
with an initial phase which reflects renal filtration (elim-
ination half-life 2–6 h), and a subsequent prolonged
phase (elimination half-life of 36 h), which represents
equilibration of the drug from tissue distribution sites.
The prolonged phase does not contribute to drug accu-
mulation following repeated administration, but it is
thought to be of pharmacological significance in medi-
ating the effects of the drug [9, 10].
Various studies have shown a correlation bet-
ween the plasma enalaprilat concentration and the inhi-
bition of ACE activity [11, 12, 13], as well as the
W. Ribeiro · M. N. Muscará · H. Moreno Jr · G. B. Mendes 
G. de Nucci (� )
Miguel Servet Clinical Pharmacology Unit,
Department of Pharmacology, Faculty of Medical Sciences,
UNICAMP, P.O. Box 6111, 13084-100, Campinas, SP, Brazil
A. R. Martins
Department of Pharmacology, Faculty of Medicine of
Ribeirão Preto, University of São Paulo, Ribeirão Preto,
SP, Brazil
Concentrações (médias) de enalapril em função do tempo mensuradas em 18 
voluntários sadios após a administração oral de 20 mg de maleato de enalapril
6
4
Farmacocinética Biodisponibilidade
ASC
7
ti
ti+1
Ci
Ci+1
Áreatrapézio = (B + b) x h / 2
Ti = (Ci+1 + Ci) x (ti+1 – ti) / 2
ASC[t1-tn] = 𝚺 Ti
i=1
n-1
8
5
ASC[0-∞] = ASC[0-tn] + ASC[tn- ∞] = 𝚺 Ti + ASC[tn- ∞]
i=1
n-1
C(t) . dt∫
tn
∞
Biodisponibilidade
[Enalapril] (ng/ml) = 366 . e–0,18 . t
A. e-k . t . dt∫
tn
∞
9
C(t) = A . e–k . t
ln C2 – ln C1
t2 – t1
= k
k = tg 𝜽
𝜽
C0
C(t) = C0 . e–k . t
10
6
C0 . e-k . t . dt∫
tn
∞
= lim C0 . e-k . t . dt∫
tn
a
a →∞
Integral imprópria
= lim C0 . (-1 / k) . e-k . t 
tn
a
a →∞
= lim C0 . (-1 / k) . (e-k . a - e-k . tn)
a →∞
= C0 . (1 / k) . lim (e-k . tn - e-k . a)
a →∞
= C0 . (1 / k) . (e-k . tn – 0)
= (1 / k) . C0 . e-k . tn = (1 / k) . Cn = ASC[tn- ∞]
ASC[0-∞] = ASC[0-tn] + ASC[tn- ∞] = 𝚺 Ti + (1 / k) . Cn
i=1
n-1
11
Problemas...
12
7
Equivalência
Ø Biodisponibilidade comparativa (estudos de 
bioequivalência) em humanos;
Ø estudos comparativos de farmacodinâmica em 
humanos;
Ø testes de dissolução in vitro.
13
Origem do problema:
Ø Fármacos pouco solúveis apresentam problemas de bioequivalência 
(ex. glibenclamida, nifedipina, ciclosporina );
Ø Estes fármacos requerem formulações galênicas otimizadas.
Conceito:
Ø A liberação do fármaco a partir da forma farmacêutica é afetada pela 
solubilidade do composto.
Ø A absorção do fármaco depende da sua permeabilidade através de 
membranes.
Sistema de classificação biofarmacêutica
14
8
Procedimento:
Ø Os fármacos são classificados com base na sua:
Ø solubilidade (máxima dosagem em 250 ml de tampão pH 1,0 a 
6,8;
Ø permeabilidade (ex., >90% de absorção).
Sistema de classificação biofarmacêutica
Classe Solubilidade Permeabilidade
I alta alta
II baixa alta
III alta baixa
IV baixa baixa
15
Dissolution determined bioavailability
q Example: glibenclamide 3.5 mg “IR” products
... dissolution ... bioavailability
q Findings, conclusion
- significant (dissolution) differences in vitro ...
- ... were also observed in vivo
- glibenclamide BA "controlled" by dissolution ...
0
50
100
150
0 3 6 9 12
0
30
60
90
0 10 20 30 40 50 60
D [%]
[min]
[ng/ml]
[h]
SocraTec R & D
16
9
Permeation determined bioavailability
q Example: ranitidin IR tablets [J. Polli, 1997]
... dissolution [water] ... bioavailability [n = 14]
q Conclusions
- significant differences in vitro ...
- ... were not confirmed in vivo
- BA not affected by drug formulation properties ... ?
0
20
40
60
80
100
0 15 30 45
D [%]
[min]
0
200
400
600
800
1000
1200
0 5 10 15 20 25
[ng/ml]
[h]
17
Biodisponibilidade relativa
Comparação da biodisponibilidade de duas formulações
Formulação teste vs. formulação de referência
and 44.2 (4.3) U· l�1in the Eupressin and Renitec, tri-
als, respectively. No differences were observed in the
basal serum ACE values between the two periods,
showing no “carry-over” effect.
The mean values of the inhibition of serum ACE
activity plotted as a function of the mean serum
enalaprilat concentrations are shown in Fig. 3. A sin-
gle binding site Michaelis-Menten type relationship
described this relation of the form Y = Ymax ×
[X]/(I50+[X]), where Y is % ACE inhibition; [X] is
enalaprilat concentration (in ng· ml�1), I50 is enalapri-
lat concentration causing 50% ACE inhibition, and
Ymax is the maximum attainable ACE inhibition with
respect to the basal value. The values obtained from
the curve by non-linear regression were I50 = 3.8 (0.2)
ng· ml�1 and Ymax: 94.7 (0.7)% (r : 0.997; P < 0.05).
The Lineweaver-Burke double reciprocal plot of the
experimental values confirmed the suitability of the
mathematical model (see insert in Fig. 3).
Discuss ion
The US Food and Drug Administration (US FDA) has
stated that the bioavailability of a given pharmaceuti-
cal formulation in vivo can be determined by mea-
surement of the active drug ingredient, therapeutic
moiety or metabolities in biological fluids as a func-
tion of time, or by quantification of an appropriate
acute pharmacological effect [20].
When enalaprilat concentration-time curves (Fig.1a)
were employed to assess bioequivalence, no significant
difference in the extent of absorption was observed
between the two formulations (assessed by inclusion of
the 90% confidence interval for individual AUC[0–24]
ratios in the bioequivalence range of 80–125%). As
the 90% confidence interval for individual Cmax ratios
was wider than the bioequivalence range, bioequiva-
lence could not be concluded for the rate of absorp-
tion, even though individual tmax differences were
402
Fig. 1 Serum enalaprilat (a)
and total enalapril (b)
concentrations (mean with
SEM) versus time curves in 18
healthy male volunteers after a
single dose (2 × 10 mg) of each
enalapril maleate tablet
formulation
Eur J Clin Pharmacol (1996) 50 : 399–405 © Springer-Verlag 1996
P H A R M AC O K I N E T I C S A N D D IS P O S I T I O N
W. Ribeiro · M. N. Muscará · A. R. Martins 
H. Moreno Jr · G. B. Mendes · G. de Nucci
Bioequivalence s tudy of two enalapr i l maleate tablet formulat ions
in healthy male volunteers
Pharmacok inet ic versus pharmacodynamic approach
Received: 29 May 1995 / Accepted in revised form: 30 October 1995
Abstract Objective: Two different conventional release
enalapril maleate tablet formulations were evaluated
for their relative bioavailability (Eupressin tablets
10 mg, Biosintética as the test formulation vs Renitec
tablets 10 mg Merck Sharp & Dhome, as the reference
formulation). A single 20 mg oral dose of each prepa-
ration was administered to 18 healthy male adult vol-
unteers and their bioequivalence was assessed by
comparing the serum enalaprilat and total enalapril
(enalaprilat plus enalapril maleate) concentration-time
curves. Angiotensin converting enzyme (ACE) activity
was also quantified in each serum sample.
Results: The pharmacokinetic parameters obtained for
each formulation were the area under the time-
concentration curve from 0 to 24 h (AUC[0–24]), max-
imum concentration Cmax and the time at which it
occurred (tmax). When serum enalaprilat concentration-
time curves were employed to assess bioequivalence,
the formulations were bioequivalent in the extent but
not in the rate of absorption. However, no difference
in either the extent or the rate of absorption were
observed when serum total enalapril vs time curves
were analysed. ACE activity-time curves were similar
for both formulations and showed that ACE was 90%
inhibited 3–5 h after enalapril administration, and till
approximately 50% after 24 h. At that time, circulat-
ing enalaprilat and total enalapril levels were less than
the tenth of Cmax. 
Conclusion: The results show that complete bioequiv-
alence of the two formulations can be concluded from
serum total enalapril concentration data, and that
serum ACE activity is not a suitable pharmacodynamic
variable for assessing bioequivalence.
Key words Bioavailability, Enalapril formulations;
pharmacokinetics, ACE-activity, human
Introduct ion
Enalaprilat (MK422, N-[(S)-1-carboxy-3-phenyl-
propyl]-L-alanyl-L-proline) is a potent angiotensin
converting enzyme (ACE) inhibitor, widely used in
essential and renovascular hypertension [1, 2]. It
reduces the plasma levels of angiotensin II and aldo-
sterone [3, 4, 5] and increases endogenous bradykinin
concentrations [6, 7].
Enalaprilat is sold as the maleate ester (MK421,
enalapril), which has enhanced gastrointestinal absorp-
tion [2]. About 60% of a dose of enalapril is absorbed
after oral administration. The compound itself is not
a potent ACE inhibitor but its subsequent in vivo
hydrolysis in the liver results in production of the active
diacid form, enalaprilat, which itself is poorly absorbed
[1, 8]. The peak plasma concentration of the pro-drug
is generally observed about 1 h after oral administra-
tion, whereas the Cmax of enalaprilat occurs after 2–4 h
[2, 9, 10].
A significant proportion of enalaprilat (43%) is elim-
inated in the urine [11, 12, 13]. Elimination is biphasic,
with an initial phase which reflects renal filtration (elim-
ination half-life 2–6 h), and a subsequent prolonged
phase (elimination half-life of 36 h), which represents
equilibration of the drug from tissue distribution sites.
The prolonged phase does not contribute to drug accu-
mulation following repeated administration, but it is
thought to be of pharmacological significance in medi-
ating the effects of the drug [9, 10].
Various studies have shown a correlation bet-
ween the plasma enalaprilat concentration and the inhi-
bition of ACE activity [11, 12, 13], as well as the
W. Ribeiro · M. N. Muscará · H. Moreno Jr · G. B. Mendes 
G. de Nucci (� )
Miguel Servet Clinical Pharmacology Unit,
Department of Pharmacology, Faculty of Medical Sciences,
UNICAMP, P.O. Box 6111, 13084-100, Campinas, SP, Brazil
A. R. Martins
Department of Pharmacology, Faculty of Medicine of
Ribeirão Preto, University of São Paulo, Ribeirão Preto,
SP, Brazil
R =
ASCteste
ASCref.
0,80 ≤ IC90% ≤ 1,25 ⟹ as formulações são BIOEQUIVALENTES
18
10
0 3 6 9 12
C
on
ce
nt
ra
tio
n
8
6
4
2
0
Hours
Cmax
tmax
19
Concentração plasmática de sertralina em 
função do tempo (média de 24 voluntários)
0 2 4 6 8 10 12 14 16 18
0
10
20
30
Zoloft (medicamento referência)
Tolrest (medicamento similar)
24 48 72 96
ng
/m
L
Horas
20
11
Análise estatística de bioequivalência
TOL/ZOL
Análise Estatística
Média Geométrica 90% IC
AUC(0-inf) 94.6 89.3 – 100.3
Cmax 99.9 91.73 – 108.8
21
Tempo (h)
D
ic
lo
fe
na
c 
(n
g/
m
l)
0:00 2:00 4:00 6:00 8:00 10:00
0
500
1000
12:00 24:00
-Sucralfate+Sucralfate
22
12
Design and analysis of 
average bioequivalence 
studies
23
Design
2x2 cross-over
washout to avoid carry-over effects
also in food-interaction
drug-drug-interaction studies may consider 
“one-sequence cross-over” or parallel 
group designs
Sequence Period 1 Period 2
1 Reference Test
2 Test Reference
24
13
Bioequivalence study using 
two parallel groups
+ study can be completed quickly, no 
washout necessary
- analysis based on inter-subject 
variability, which is generally much 
higher than intra-subject variability: much 
more subjects needed
25
Situations in which a parallel 
design may be appropriate
long half-life (® long washout necessary in a 
crossover study)
subjects are very ill patients
high drop-out rate per period 
cost of increasing the number of subjects is 
much less than that of adding an additional 
period
26
14
Criteria
Mean (AUCtest / AUCref) ⊂ [0,80 – 1,25]
Mean (Cmaxtest / Cmaxref) ⊂ [0,80 – 1,25]
27
FDA EMEA CANADÁ BRASIL ICH
População
Idade > 18 anos 18-55 anos 18-50 anos
Sexo ambos ambos ambos
Restrições Desenho do Estudo
Nº de voluntários>= 12 >= 12 >= 12 ±24
WashOut (nº 1/2 vidas) 5 3 6 ?? 7
Nº de pontos de coleta 12 a 18 > 80% AUCinf
Nº de pontos fase terminal 3 a 4
Nº de 1/2 vidas de coleta > 3 > 3 3
Opções de coleta até 72h *
Divisão em sub-grupos não acons.
Conduta Terapêutica
Água com a dose 240 mL >150 mL
Apresentação a utilizar maior dose segurança
Múltiplos comprimidos permite
Restrições / Crit. De Exclusão
Fumo < 20 cig/dia < 10 cig/dia evitar
Alcool 24h antes
Peso não especifica BMI ±15% normal
"Tônica das restrições" safety
- 21 CFR part 320
- Guidance: Bioavailability & Bioequivalence 
Studies for orally administered drug products - 
FDA Oct/2000
Note for Guidance on the 
Investigation of Bioavailability 
and Bioequivalence - EMEA - 
CPMP - 2001
Resolução RDC 84/02 e anexos
28
15
FDA EMEA CANADÁ BRASIL ICH
Dieta
Jejum Pré-dose ? noite anterior
Jejum Pós-dose 4 horas ?
Interrupção de Líquidos 1h antes
Liberação de Líquidos 1h depois
Padronização da Dieta sim sim
Documentação
Período de guarda 5 anos
Outras questões
Estudo piloto previsto
Monitoria do estudo previsto
Repetição do Estudo x CEP ?
Parent Comp. X Metabolite Parent Parent/Metabolite
- 21 CFR part 320
- Guidance: Bioavailability & Bioequivalence 
Studies for orally administered drug products - 
FDA Oct/2000
Note for Guidance on the 
Investigation of Bioavailability 
and Bioequivalence - EMEA - 
CPMP - 2001
Resolução RDC 84/02 e anexos
29
Metodologias para análise de fármacos
 Ensaios biológicos.
 Testes colorimétricos.
 Imunoensaios.
 Espectrofotometria (uv visível, i.v, fluorescência).
 Espectrometria de massa.
 Ressonância magnética nuclear.
 Cromatografia de camada delgada.
 Cromatografia gasosa.
 Cromatografia líquida de alta pressão
 LC-MS-MS
30
16
Metodologia para dosagem em 
líquidos biológicos
H.P.L.C.
H.P.L.C. acoplado a MS (LC-MS-MS).
31
Analytical variables
1.1) Internal Standard
Similar structure (Analogues)
Deuterated isotopes
1.2) Ionization Methods 
Positive Electrospray (ES+)
Negative Electrospray (ES-)
Atmosphere Pressure Chemical Ionization (APCI)
1.3) Sample Extraction
Liquid/Liquid
Solid Phase Extraction (SPE)
32
17
Analytical column: Genesis C18, 4µm (150 mm x 4.6 mm i.d.)
Mobile phase: 50% Acetonitrile/50% Water & 10mM formic acid
Ionization mode: Positive electrospray (ES+)
Ions were monitored by MRM (Multiple Reaction Monitoring)
Sample Extraction: Solid/Liquid (Oasis SPE cartridges)
Determination of Amoxicilin using Cefadroxil as 
internal standard
33
Chemical Structures
N
SN
O
H
HO
NH2
 HO
H
CH3
CH3 
COOH
Amoxicilin
M.W. 365.40 g/mol
Cefadroxil
M.W. 363.38 g/mol
N
N
O
H
HO
NH2
 HO
H
S
COOH
CH3
34
18
m/z 349
m/z 364
N
N
O
H
HO
NH2
 HO
H
S
COOH
CH3
H
m/z 208
Fragmentation routes for 
Amoxicilin and Cefadroxil
m/z 366
H
N
SN
O
H
HO
NH2
 HO
H
CH3
CH3 
O
OH
35
Mass Spectra - Amoxicilin
100 150 200 250 300 350 400
Da/e0
100
%
0
100
%
Daughters of 366
348.9
113.7
208.2
159.9 304.7234.4 276.7 330.6
366.2
366.3
349.0
104.4
388.0
36
19
100 200 300 400 500 600
M/z0
100
%
0
100
%
Daughters of 364
208.0
157.9 347.1
364.0
Mass Spectra - Cefadroxil
37
Amoxicillin and Cefadroxil chromatograms
Amoxicilin 0.05 µg/mL
0
100
%
366.00 > 349.202.63
Cefadroxil 0.05 µg/mL
1.00 2.00 3.00 4.00 5.00
Time (min)0
100
%
364.00 > 207.80
2.63
38
20
Amoxicillin Calibration Curve
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0
µg/mL0
3.49
Response
39
Total run time: 4.0 min.
LOQ: 50 ng/mL 
Precision: 15.4 %
Accuracy: - 6.5 %
40
21
Analytical column: Genesis C18, 4µm (150 mm x 4.6 mm 
i.d.)
Mobile phase: 50% acetonitrile in 10 mM acetic acid + 20 
mM ammonium acetate aqueous solution
Ionization mode: Positive electrospray (ES+)
Ions were monitored by MRM (Multiple Reaction 
Monitoring)
Sample Extraction: Solid/Liquid (Oasis SPE cartridges)
Determination of Enalaprilat using 
Enalaprilat-phenyl-D5 as internal standard
41
Chemical Structures
N
N
O
HOOC
H
CH3
COOH
Enalaprilat
M.W. 348.39 g/mol
Enalaprilat-phenyl-D5
M.W. 353.42 g/mol
N
N
O
HOOC
H
CH3
COOH
D
D
D
D
D
42
22
m/z 211m/z 206
m/z 349
N
N
O
HOOC
H
CH3
COOH
H
m/z 354
N
N
O
HOOC
H
CH3
COOH
D
D
D
D
D
H
Fragmentation routes for 
Enalaprilat and Enalaprilat-phenyl-D5
43
Enalaprilat Mass Spectra
50 100 150 200 250 300 350 400 450 500
Da/e0
100
%
0
100
%
Daughters of 349 
206
160102 116 303
349
Scan ES+ 
349
64 81 20691 185 303214
350
371
415 429
44
23
Calibration Curve
Response
50.0 100.0 150.0 200.0
ng/mL
0
12.9
45
Total run time: 4.0 min.
LOQ: 0.5 ng/mL
Precision: 6.9 %
Accuracy: 2.8 %
46
24
Sample Extraction
Liquid/Liquid
two phases (aqueous/organic solvent)
faster
cheaper
SPE (Solid Phase Extraction) cartridges 
Easier for automatization
47
Typical procedure for liquid/liquid extraction
1. Dispense the sample into appropriate tubes;
2. Dispense the corresponding I.S. and vortex-
mix the samples for approximately 10 s.;
3. Add the organic solvent mixture (e.g. diethyl-
ether/hexane 80:20) and vortex-mix the samples 
for 40 s.
4. Centrifugation.
48
25
5. Remove the upper organic layer and transfer 
it to clean test tubes;
6. Remove the solvent using a flow of N2 at 
37oC;
7. Add mobile phase and vortex-mix for 15s to 
reconstitute the residues;
8. Transfer the solution to vials.
49
Typical procedure for Solid Phase Extraction 
(SPE) cartridges
50
26
Some examples of use in bioequivalence studies
Liquid/Liquid
Cyproterone
Fluconazole
Captopril
Omeprazole
Paroxetine
Roxithromycin
SPE
Cephalexin
Cefadroxil
Amoxicilin
Didanosine
51
Conclusions
Ø LC-MS-MS is a very suitable technique for 
quantification of drugs in biological fluids for 
bioequivalence studies, since it presents:
Ø Sensitivity
Ø Selectivity
Ø Through-put
Ø Robust
Ø Low complexity
52
27
Obrigado pela atenção!
53