Quantification of cyproheptadine in human

plasma by highperformance liquid

chromatography coupled to electrospray
tandem mass spectrometry in a
bioequivalence study
Gustavo Duarte Mendes
a,b,c
*, Andr Arruda
a
, Lu Shi Chen
b
,
Jos Cssio de Almeida Magalhes
a,c
, Khalid M Alkharfy
d
and
Gilberto De Nucci
a,d
ABSTRACT: A rapid, sensitive and specific method to quantify cyproheptadine in human plasma using amitriptyline as the
internal standard (IS) is described. The analyte and the IS were extracted from plasma by liquidliquid extraction using a
diethylether/dichloromethane (70/30; v/v) solvent. After removing and drying the organic phase, the extracts were
reconstituted with a fixed volume of acetonitrile/water (50/50 v/v) +0.1% of acetic acid. The extracts were analyzed by high
performance liquid chromatography coupled to electrospray tandem mass spectrometry (LCMS/MS). Chromatography was
performed isocratically using an Alltech Prevail C18 5 m analytical column, (150 mmx 4.6mm I.D.). The method had a
chromatographic run time of 4 min and a linear calibration curve ranging from 0.05 to 10 ng/mL (r2 >0.99). The limit of
quantification was 0.05 ng/mL. This HPLC/MS/MS procedure was used to assess the bioequivalence of cyproheptadine in two
cyproheptadine +cobamamide (4 mg+1 mg) tablet formulations (Cobactin [cyproheptadine +cobamamide] test formulation
supplied fromZambon Laboratrios Farmacuticos Ltda. and Cobavital fromSolvay Farma (standard reference formulation)).
A single 4 mg+1 mg [cyproheptadine +cobamamide] dose of each formulation was administered to healthy volunteers. The
study was conducted using an open, randomized, twoperiod crossover design with a 1week washout interval. Since the 90%
CI for Cmax and AUCs ratios were all within the 80125% bioequivalence limit proposed by the US Food and Drug
Administration, it was concluded that the cyproheptadine test formulation (Cobactin) is bioequivalent to the Cobavital
formulation for both the rate and the extent of absorption of cyproheptadine. Copyright 2011 John Wiley & Sons, Ltd.
Keywords: cyproheptadine; healthy volunteer; plasma; pharmacokinetic; LCMS/MS
Introduction
Cyproheptadine hydrochloride (CAS no. 969335) is an antihista-
minic and antiserotonergic agent with inhibitory activities for
ltype calcium channels (Gunja et al., 2004; Yamamoto et al., 2006;
Fes et al., 2009a, 2009b). It has anticholinergic andsedative effects.
It is indicated for perennial allergic and vasomotor rhinitis, allergic
conjunctivitis, mild and cold urticaria, and is a proven migraine
prophylaxis, an appetite enhancer and, in adrenocorticotropic
hormonedependent Cushings syndrome, it normalizes cortisol
indexes (Watemberg et al., 1999). The colateral effects
of cyproheptadine are drowsiness, coordination disturbances,
increased appetite, weight gain, exacerbation of depression, dry
mouth and urinary retention (Watemberg et al., 1999). After an oral
dose of 8 mg of cyproheptadine, the C
max
, AUC and T
max
for total
cyproheptadine were 30 ng/mL, 206 ng h/mL and 4h, respectively
(Gunja et al., 2004). Cyproheptadine has been determined in
human plasma by CGMS (Hasegawa et al., 2006), in human serum
by LCMS (Gunja et al., 2004), in animal urine by GLC (Hucker and
Hutt, 1983), LCMSMS (Fes et al., 2009a; Fente et al., 2009) and
reversedphaseHPLC (Kountourellis and Ebete, 1995), and in
pharmaceutical syrup samples by LCMS/MS (Fes et al., 2009b).
Here we describe a fast, sensitive and selective method for
measuring plasma cyproheptadine using liquid chromatography
* Correspondence to: G. D. Mendes, Department of Pharmacology, State
University of Campinas, Campinas, Brazil. Email: gugamendes@terra.com.br
a
Department of Pharmacology, State University of Campinas, Campinas,
Brazil
b
Galeno Research Unit, Campinas, Brazil
c
Faculty of Odontology, University Camilo Castelo Branco (UNICASTELO),
So Paulo, Brazil
d
Department of Clinical Pharmacy, College of Pharmacy, King Saud
University, Riyadh, Saudi Arabia
Abbreviations used: ANVISA, Brazilian National Sanitary Surveillance
Agency; CAS, Chemical Abstract Service; FDA, Food and Drug Administration.
Biomed. Chromatogr. 2012; 26: 129136 Copyright 2011 John Wiley & Sons, Ltd.
Short communication
Received 15 January 2011, Accepted 19 January 2011 Published online in Wiley Online Library: 23 March 2011
(wileyonlinelibrary.com) DOI 10.1002/bmc.1618
1
2
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coupled to tandem mass spectrometry (LCMSMS) with positive
ion electrospray ionization using amitriptyline as the internal
standard(IS, Fig. 1). This methodwas employedina bioequivalence
study of cyproheptadine in two cyproheptadine+cobamamide
(4 +1 mg) tablet formulations. The bioequivalence study was
conducted in 40 healthy volunteers using a singledose, twoway,
open, randomized crossover design with 1week washout period
between the doses.
Experimental
Chemicals and reagents
Cyproheptadine hydrochloride (lot G, current lot) was provided by USP.
Amitriptyline hydrochloride was provided by Honeywell (AMPT/0604014,
March/2011). Acetonitrile, methanol, formic acid, glacial acetic acid and
ammonium formiate were purchased from J. T. Baker (Phillipsburg, NJ,
USA). Diethylether and dichloromethane were supplied by Mallinckrodt
(Phillipsburg, NJ, USA). All chemicals and solvent were of analytical
reagentgrade except for acetonitrile and methanol, which were HPLC
grade. A milliQ Gradient A10 water purification system from Millipore
(Bedford, MA, USA) was used. Blank human blood was collected from
healthy drugfree volunteers into sodium heparincontaining tubes and
plasma was obtained by centrifugation. Plasma was obtained by
centrifugation of blood treated with the anticoagulant sodium heparin.
Pooled plasma was prepared and stored at approximately 20C until
needed.
Calibration standards and quality control samples
Stock solutions of cyproheptadine and internal standard (amitriptyline)
were prepared in methanolwater (50:50 v/v). Calibration curves of
cyproheptadine were prepared by spiking blank plasma at concentrations
of 0.05, 0.1, 0.2, 0.5, 1, 3, 6 and 10 ng/mL. The analysis was carried out in
duplicate for each concentration. The quality control (QC) samples were
prepared in blank plasma at concentrations of 0.15, 1.5 and 8 ng/mL (QCA,
QCB and QCC, respectively). For each analytical batch, the spiked plasma
samples (standards and quality controls) were extracted along with the
unknown samples. All lots of blank plasma used in analytical batches
during the validation process and volunteer samples quantification
(calibration standards and QCs) were previously tested and pooled,
fractioned (50 mL flasks) and stored at 20C until use.
Liquid chromatography and mass spectrometry conditions
An aliquot of each plasma extract was injected into an Alltech Prevail C
18
5 m analytical column (1504.6 mm i.d.) operating at 400C. The
compounds were eluted by pumping the mobile phase [acetonitrilewater
Figure 1. Fullscan mass spectra in (A) trace and product ion spectra in (B) trace of (1) cyproheptadine and (2) amitriptyline.
G. D. Mendes et al.
Biomed. Chromatogr. 2012; 26: 129136 Copyright 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc
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(80/20; v/v) +0.1%formic acid+5 mM ammoniumformiate] at a flowrate of
1.2 mL/min. Under these conditions, typical retention times were
2.70.3min for cyproheptadine and 2.90.3min for amitriptyline, and
backpressure values of approximately 70bar were observed. A split of the
column eluant of approximately 1:2.5 was included. The temperature of the
autosampler was kept at 12C and the runtime was 4min. The mass
spectrometer (API 4000 Sciex/Applied Biosystems, Canada) equipped with
an electrospray source using a cross flowcounter electrode run in a positive
mode (ES+), was set up in multiple reaction monitoring (MRM), monitoring
the transitions 288.30 96.20 and 278.30 91.10 for cyproheptadine and
IS, respectively. Figure 1 shows the fullscan spectra (upper trace) and the
product ion spectra (lower trace) obtained for cyproheptadine and
amitriptyline. In order to optimize all the MS parameters, a standard
solution of the analyte and IS was infused into the mass spectrometer. The
source block temperaturewas set at 500Candthe turboionspray voltage to
5.5kV. Nitrogen was used as collision gas. For cyproheptadine, the
declustering potential, collision energy and collision exit potential were
76V, 35 eV and 8V, respectively. The corresponding values for IS were 71V,
33eV and 8V, respectively. Data were acquired by Analyst software (version
1.4.1, Applied Biosystems, Foster City, CA, USA).
Sample preparation
Twohundred microliters of sample human plasma were pipetted into
glass tubes followed by 50 L of the internal standard solution (50 ng/mL
of amitriptyline in methanolwater, 50:50 v/v solution). The samples were
vortexmixed for approximately 10 s. Diethyletherdichlorometane
(70:30 v/v) was added (4 mL) to all tubes, which were then vortexmixed
for 40 s. The samples were centrifuged at 4000 rpm for 4 min at 4C. The
tubes were frozen for 15 min at 80C. The upper organic phase was
transferred to another set of clean glass tubes and evaporated to dryness
under N2 at 40C. The dry residues were dissolved with 0.2 mL of a
solution of acetonitrilewater (50:50 v/v) +0.1% of acetic acid, vortex
mixed for 10 s to reconstitute the residues and transferred to 96well
plates using automatic pipettes with disposable plastic tips.
Bioanalytical method validation
The method validation assays were carried out according to the United
States Food and Drug Administration (FDA) bioanalytical method
validation guidance (Food and Drug Administration, 2001) and the
Brazilian National Sanitary Surveillance Agency (ANVISARE 899, 2003).
Three validation batches were prepared on different days by spiking
blank plasma with the working solutions to produce the standard curve
points and QCs. The extraction procedure was the same as described
above. Each batch was constituted of duplicates of blank (processed
without the IS) and zero plasma samples (blank with IS and processed)
to ensure the absence of interferences, followed by the standard
samples (duplicates of 0.05, 0.1, 0.2, 0.5, 1, 3, 6 and 10 ng/mL
of cyproheptadine) and eight replicates of the the lower limit
of quantification (LLOQ, 0.05 ng/mL) and QC samples, with concentra-
tions equivalent to 0.15 ng/mL (low level), 1.5 ng/mL (medium level) and
8 ng/mL (high level).
Calibration curves were generated by using the ratios of the analyte
peak area to the IS peak area vs analyte concentration and were fitted to
the linear equation ( y =a +bx) by weighted (factor: 1/x
2
) least squares
linearity regression with R >0.98. The limit of detection and the LLOQ
were determined, calculated as the concentrations with a signaltonoise
ratio of 3 and 10, respectively. Each backcalculated concentration
standard should meet the following acceptable criteria: no more than
20% deviation at LLOQ and no more than 15% deviation above LLOQ.
Betweenrun and withinrun accuracy and precision were calculated
for LLOQ and QCs levels, considering eight measurements of each
concentration. The accuracy of the method was shown in relative values
(accuracy, %) and calculated based on the difference between the
calculated mean and nominal concentrations, whereas precision was
evaluated by calculating the within and betweenrun relative standard
deviations (RSD%).
Specificity. Blank plasma samples of healthy human used for testing
the specificity of the method were obtained from six different sources
(four normal, hemolyzed and lipemic).
Recovery. The absolute extraction recovery of cyproheptadine and IS
was assessed by comparing peak area ratios obtained from extracted
plasma samples with those from the standard solutions at the same
concentration. This procedure was performed using five aliquots from
three different sources of normal human plasma at each QC level (0.15,
1.5 and 8 ng/mL). The relative recovery was calculated as peak area
ratios obtained from extracted plasma samples with postextracted
spiked samples (matrix extracted standard solution) at the same
concentration at each QC level.
Stability. Quality control samples (0.15, 1.5 and 8; five measurements
of each concentration) were subjected to shortterm (8 h) at room
temperature and 48 h postprocessing (12C) stability tests. Test samples
were analyzed and the found cyproheptadine concentrations were
compared with freshly prepared samples. Stock and working solutions
stability assays were processed by comparing fresh solutions with
previously prepared ones. All stability data were reported as relative
errors (RE, %).
Employment of the bioanalytical method
Volunteers samples were quantified into analytical batches constituted
by calibration curve standards (in duplicate) followed by one of each QC
sample (low, middle and high) between every other 10 volunteer
samples. For each analytical batch, a convenient quantity of pooled
blank plasma was thawed to prepare fresh calibration standards and QC
samples.
Clinical protocol. The study consisted of an open study of 40 healthy
volunteers of both sexes, aged between 18 and 53 years old. Screening
assessment was performed by means of medical history, general
physical examination, electrocardiogram and clinical laboratory tests.
After an washout period of at least 2 weeks, the individuals who
qualified were confined for two periods of approximately 36 h. Each
confinement was intervaled by a period of 1 week. During confinement,
a 7 mL blood sample was collected before dosing and 0.25, 0.5, 0.75, 1,
1.33, 1.67, 2, 2.33, 2.67, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 24,
48, 72 and 96 h postdosing. A general physical examination,
electrocardiogram and clinical laboratory tests were also performed
during the poststudy period. Blood samples were centrifuged at
approximately 2000g for 4 min at room temperature and the plasma was
stored at 20C until assayed for cyproheptadine content.
Formulations. The following formulations were employed: Cobactin
(test formulation manufactured by Eurofarma Laboratrios Ltda, Brazil
and distributed by Zambon Laboratrios Farmacuticos, Brazil; lot no.
56407) and Cobavital (standard reference formulation from Solvay
Farma Ltda, lot no. 801374).
Pharmacokinetics and statistical analysis. Bioequivalence between
the two formulations was assessed by calculating individual test/
reference ratios for the peak of cyproheptadine concentration (C
max
),
area under curve (AUC) of plasma concentration until the last
concentration observed (AUC
last
), and the area under curve between
the first sample (predosage) and infinite (AUC
0inf
). The C
max
and the
time taken to achieve this concentration (T
max
) were obtained directly
from the curves. The areas under the cyproheptadine plasma
concentration vs time curves from 0 to the last detectable concentration
(AUC
last
) were calculated by applying the linear trapezoid rule.
Extrapolation of these areas to infinity (AUC
0inf
) was done by adding
the value C
last
/k
e
to the calculated AUC
last
(where C
last
=the last
detectable concentration). The AUC and C
max
data for the two
formulations were analyzed by ANOVA to establish whether the 90%
confidence interval (CI) of the ratios was within the 80125% interval
indicating bioequivalence as proposed by FDA and ANVISA. Parametric
and nonparametric analyses of lntransformed arithmetic means and
Quantification of cyproheptadine in human plasma by LCMSMS
Biomed. Chromatogr. 2012; 26: 129136 Copyright 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc
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individual T
max
differences between test and reference formulations
were also performed.
Results and discussion
Method development
Analytical methods employed for the quantitative determina-
tion of drugs and their metabolites in biological samples must
generate reproducible and reliable data in order to permit valid
interpretation of the studies they support (Stokvis et al., 2005a;
Marzo and Dal Bo, 2006; Rozet et al., 2007; Shah, 2007; Xu et al.,
2007). During the validation process and routine methodology
employment, the internal standard plays an important role in
achieving satisfactory accuracy and precision(Stokvis et al., 2005b).
In this procedure, amitriptyline was adopted as the internal
standard because its structure, retention action and ionization
as well as extraction efficiency are similar to those of
cyproheptadine (Fig. 1). The mass transitions to set mass
spectrometry detection (MRM) were obtained from full and
product ion scans in positive mode by infusion of cyprohepta-
dine and amitriptyline working solution. Analyte and IS showed
precursor ions ([M+H]
+
) at m/z =288.30 and m/z =278.30,
respectively. Precursor ions were submitted to collisioninduced
Figure 2. MRM chromatogram of a LLOQ (0.05 ng/mL) sample: (A) cyproheptadine channel and (B) I.S. (amitriptyline) channel. MRM chromatograms
of blank normal human plasma (C) cyproheptadine and (D) amitriptyline.
G. D. Mendes et al.
Biomed. Chromatogr. 2012; 26: 129136 Copyright 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc
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dissociation (Fig. 2). All settings were similar for analyte and IS
due to their analogue structures and ionization behavior. Most
abundant product ions were m/z =96.20 (for cyproheptadine)
and m/z =91.10 (for amitriptyline). The proposed fragmentation
pathways are illustrated in Fig. 1.
For liquid chromatography an Alltech Prevail C
18
5 m
analytical column (150 mm4.6 mm i.d.) was used, operated
at 40C with methanolwater (80:20; v/v) +0.1% mM formic
acid + 5 mM ammonium formiate as mobile phase, which
produced fast and reproducible separation. Both cyprohepta-
dine and IS peaks presented convenient (after void) retention
times (2.7 and 2.9 min, respectively). To obtain a clean
chromatogram and achieve a sufficient extraction recovery,
liquidliquid extraction (LLE) was used and several solvents
mixtures and additives were investigated (Xu et al., 2007). The
mixture of diethyletherhexane (70:30; v/v) was eventually
proved to be the best in terms of the higher extraction recovery
and absences of endogenous interference at the retention time
of cyproheptadine and amitriptyline chromatogram.
Bioanalytical method validation and performance
Following development of a bioanalytical LCMS/MS assay and
before implementation into bioanalytical assays and routine use,
it needs to be validated. Validation is essential to ensure the
acquired data are accurate (Rosing et al., 2000). All validation
criteria (specificity, linearity, recovery, accuracy and precision)
were evaluated to assess the methods performance. In order to
check the specificity/selectivity of the method, six plasma lots
from different sources (four normal plasma, lipemic and
hemolized) were tested. The simplest regression method for
the calibration curves of the cyproheptadine was y =a +bx from
0.05 to 10 ng/mL (calibration curve y =0.0725x +0.000515,
r =0.9978).
The recoveries of cyproheptadine were 93.3, 83.2 and 93.1%
for the low, medium and high pools, respectively. The recovery
of the IS tested at the concentration used in the study samples
was 83.0%. No significant matrix effect was observed. The
validated LLOQ was 0.05 ng/mL, defined as the lowest
concentration at which both the precision and accuracy were
20%. Within and betweenrun precision and accuracy for the
LLOQ and QCs are summarized in Table 1. Stability tests
performed indicated no significant degradation under the
conditions described (Tables 25).
As shown in Fig. 2, no endogenous peak was observed in the
mass chromatogram of blank plasma. The chromatogram for
the standard LLOQ sample is shown in Fig. 2, in which the
retention times for cyproheptadine and IS were 2.7 (0.3) and
Table 1. Accuracy and precision data for cyproheptadine from the prestudy validation in human plasma
Nominal concentration (ng/mL) 0.05 0.15 1.5 8.0
Intrabatch, N=7
Mean range 0.049 0.150 1.49 8.7
0.0390.055 0.1390.155 1.441.53 8.39.1
Precision (%) 10.10% 3.65% 2.26% 3.16%
Accuracy (%) 97.94% 99.81% 99.33% 108.92%
Interbatch, N=21
Mean range 0.048 0.151 1.53 8.8
0.0390.055 0.1240.163 1.341.65 8.29.5
Precision (%) 8.29% 5.91% 4.93% 3.75%
Accuracy (%) 96.19% 100.43% 102.07% 110.02%
Table 2. Postprocessing stability test (values in ng/mL)
Reference values Values after
47 h 50 min
Reference values Values after
47 h 50 min
Reference values Values after
47 h 50 min
Low sample Medium sample High sample
Mean 0.147 0.153 1.5 1.55 8.55 8.39
CV (%) 2.2 3.2 2.1 2.2 1.4 1.6
Variation 4.1 3.3 1.9
Table 3. Freezeandthaw stability test (values in ng/mL)
Reference values Values after
three cycles
Reference values Values after
three cycles
Reference values Values after
three cycles
Low sample Medium sample High sample
Mean 0.146 0.136 1.47 1.41 7.82 7.73
CV (%) 3.4 5.2 1.4 1.3 11.2 2.4
Variation 6.8 4.1 1.2
Quantification of cyproheptadine in human plasma by LCMSMS
Biomed. Chromatogr. 2012; 26: 129136 Copyright 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc
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2.9 (0.3) min, respectively. The mean cyproheptadine plasma
concentrations vs time profiles after a single oral dose of each
4 mg tablet formulation of cyproheptadine are shown in Fig. 3.
The simplest regression method for the calibration curves of the
cyproheptadine was y =a +bx from 0.05 to 10 ng/mL (calibration
curve y =0.0725x +0.000515, r =0.9978).
This is the first LCMSMS method developed for measuring
cyproheptadine in human plasma. Conjugated cyproheptadine
was determinedinplasma by CGMS (LLOQ0.2ng/mL, RT 7.98 min,
solid extraction, 0.1mL plasma; Hasegawa et al., 2006), in human
serum by LCMS (solid extraction; Gunja et al., 2004), in animal
urine by GLC (LLOQ 3 ng/mL, liquidliquid extraction; Hucker and
Hutt, 1983), LCMSMS (LLOQ 0.15ng/mL, RT 1020 min, solid
extraction, 2 mL urine; Fes et al., 2009a; Fente et al., 2009) and
reversedphaseHPLC (solid extraction; Kountourellis and Ebete,
1995), and in pharmaceutical syrup samples by LCMS/MS (LLOQ
1 ng/mL, RT 7.29min, liquidliquid extraction; Fes et al., 2009b).
Our method has good sensitivity (LLOQ of 0.05 ng/mL), and
can be carried out in a short time (RT of 2.7 min for
cyproheptadine), permitting a high throughput. As demonstrat-
ed in this study, the present LCMSMS method is simple and
selective for the determination of cyproheptadine in human
plasma, thus it can be used for pharmacokinetic and bioequiva-
lence studies of cyproheptadine.The advantages of the herein
reported methodology are the throughput and sensitivity of the
LCMS/MS technique along with the feasibility and excellent
performance for the bioanalytical purpose within FDA and
ANVISA criteria.
Stability
Stock and working solutions of cyproheptadine and amitripty-
line were stable for 7 days when stored at 4C (n =5, RE 1.2%)
and at room temperature (25C) for 6 h (n =5, RE 11.5%),
respectively. Cyproheptadine in human plasma (at low and high
QC concentrations) presented no significant degradation after
(n =5): postprocessing (48 h, RE =4.1 and 1.9%, respectively),
freezeandthaw (three cycles, RE =6.8 and 1.2%), shortterm
(8 h, RE =9.6 and 1.5%) and longterm (56 days, RE =1.4
and 7.5%).
Pharmacokinetic study
The whole assay was conducted strictly in accordance with the
current Good Clinical Practices. The study began with 40
healthy volunteers and finished with 38 healthy volunteers.
One volunteer dropped out of the study for personal reasons
and another owing to adverse effects (vomiting). The method
was successfully employed to determine cyproheptadine
concentrations in human plasma samples after the administra-
tion of a 4 +1 mg oral dose of cyproheptadine +cobamamide.
The observed cyproheptadine peak plasma concentration
(C
max
) value (1.25 ng/mL) differed from that reported in the
literature (30 ng/mL; Gunja et al., 2004). However, Gunja et al.
(2004) measured total cyproheptadine (conjugated and drug)
whereas our method is sensitive enough for quantification of
cyproheptadine. After the oral administration of the cyprohep-
tadine tablets to the volunteers, the observed cyproheptadine
peak plasma concentration (C
max
) values and the time values
taken to achieve (T
max
) were equivalent between the formula-
tions (Tables 6 and 7). In addition, the calculated 90% CI
for mean C
max
, AUC
last
and AUC
0inf
Cobactin/Cobavital
individual ratios were within the 80125% bioequivalence limit
defined by the US FDA and ANVISA. Supported by these data,
it is possible to state that both formulations achieved equiva-
lent bioavailability.
Table 4. Shortterm stability test (values in ng/mL)
Reference values Values after 8 h Reference values Values after 8 h Reference values Values after 8 h
Low sample Medium sample High sample
Mean 0.146 0.132 1.47 1.33 7.82 7.94
CV (%) 3.4 6.3 1.4 4.6 11.2 2.2
Variation 9.6 9.5 1.5
Table 5. Longterm stability test (values in ng/mL)
Reference values Values after
56 days
Reference values Values after
56 days
Reference values Values after
56 days
Low sample Medium sample High sample
Mean 0.146 0.144 1.6 1.43 8.98 8.31
CV (%) 6.5 11.6 2.0 5.0 1.7 4.0
Variation 1.4 10.6 7.5
Figure 3. Cyproheptadine plasma mean concentrations vs time profile
obtained after the single oral administration of 4 mg of cyproheptadine
formulation.
G. D. Mendes et al.
Biomed. Chromatogr. 2012; 26: 129136 Copyright 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc
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Conclusion
This article describes the first rapid, reliable and fully validated
bioanalytical methodology using liquid chromatography cou-
pled to tandem mass spectrometry (LCMS/MS) for quantifica-
tion of cyproheptadine pharmacokinetic and bioequivalence
studies. The achieved limit of quantification (0.05 ng/mL) was
good enough for all the pharmacokinetic parameters and the
throughput obtained allowed rapid quantification of biological
samples coming from clinical studies. Also, since all validation
parameters were in accordance with the FDA and ANVISA
guidelines, the protocol robustness was demonstrated by its
employment in a comparative bioavailability study, where more
than 2200 samples were analyzed.
The cyproheptadine relative bioavailability between both
formulations (Cobactin, test and Cobavital, reference) was
assessed by calculating individual test/reference ratios for C
max
,
AUC
last
and AUC
0inf
and pharmacokinetic profiles indicated
bioequivalence since all ratios were as proposed by FDA and
ANVISA.
Conflict of interest
The bioequivalence trial was sponsored by Zambon Laboratrios
Farmacuticos Ltda.
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Table 6. Cyproheptadine mean pharmacokinetic parameters obtained from 38 volunteers after administration of each 4 mg
cyproheptadine +1 mg cobamanide tablet formulation
Parameter Unit N Mean SD Min Median Max CV%
Reference
AUC
% extrap
(%) 38 17.98 7.42 8.29 15.23 41.36 41.30
AUC
all
(ng h/mL) 38 32.84 10.13 14.65 31.93 61.22 30.86
AUC
inf
(ng h/mL) 38 40.82 15.71 16.41 38.49 91.05 38.49
AUC
last
(ng h/mL) 38 32.71 10.32 14.65 31.93 61.22 31.55
C
last
(ng/mL) 38 0.13 0.06 0.05 0.12 0.33 48.92
C
max
(ng/mL) 38 1.25 0.41 0.59 1.28 2.21 33.03
k
e
(1/h) 38 0.02 0.01 0.01 0.02 0.03 27.39
T
1/2
(h) 38 38.21 10.70 21.46 35.86 69.15 28.02
T
last
(h) 38 91.85 9.43 72.00 96.07 97.82 10.27
T
max
(h) 38 4.68 1.32 2.67 4.50 9.00 28.15
Test
AUC
% extrap
(%) 38 20.20 12.94 8.19 15.35 62.33 64.06
AUC
all
(ng h/mL) 38 34.88 15.21 17.71 33.90 86.23 43.62
AUC
inf
(ng h/mL) 38 47.00 32.02 19.62 39.86 197.59 68.13
AUC
last
(ng h/mL) 38 34.78 15.32 16.54 33.90 86.23 44.05
C
last
(ng/mL) 38 0.16 0.14 0.06 0.12 0.70 86.92
C
max
(ng/mL) 38 1.31 0.48 0.61 1.19 2.96 37.01
k
e
(1/h) 38 0.02 0.01 0.01 0.02 0.06 46.44
T
1/2
(h) 38 41.83 22.35 11.16 35.30 128.18 53.43
T
last
(h) 38 90.67 15.22 24.00 96.11 97.67 16.78
T
max
(h) 38 4.97 1.41 2.67 5.00 8.00 28.45
Table 7. Geometric mean of individual AUC
last
, AUC
0inf
and
C
max
ratios (test/reference formulation), the respective 90%
CI and power
Percentage
geometric mean
90% CI Power
N=38
C
max
% ratio 104.43 98.14111.13 1.0000
AUC
last
% ratio 103.73 97.90109.90 1.0000
AUC
inf
% ratio 107.98 99.84116.78 0.9982
Quantification of cyproheptadine in human plasma by LCMSMS
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