Research article

Received: 31 July 2012, Revised: 1 October 2012, Accepted: 13 November 2012 Published online in Wiley Online Library: 13 December 2012

(wileyonlinelibrary.com) DOI 10.1002/bio.2467

A novel spectrofluorimetric method for the

determination of calcitonin in ampules
through derivatization with fluorescamine
Deniz Koçdan and Hasan Basan*
ABSTRACT: In this study, a simple, sensitive and selective spectroflourimetric method has been developed for the determination
of salmon calcitonin (sCT) in ampules. The method is based on the reaction between sCT and fluorescamine at pH 8.5 in borate
buffer, resulting in a highly fluorescent derivative. Fluorescence of derivatized sCT solutions was measured by setting the
excitation and emission monochromators and slit widths to 390, 484 and 10 nm, respectively. Sevaral derivatization parameters
were optimized. A calibration graph was constructed using standard solutions of the derivatized calcitonin in the range
0.5–6.0 mg/mL. Limit of detection and limit of quantification values were determined to be 0.124 and 0.372 mg/mL, respectively.
The proposed method was successfully applied for the determination of sCT in commercially available ampules. High recovery
values (101.0–102.0 %), and a low relative standard deviation (RSD %) value (5.3–5.4) proved the accuracy and precision of
the proposed method. An isocratic reversed-phase high-performance liquid chromatographic (HPLC) method, as a reference,
was also developed for the determination of sCT. A reversed-phase NucleosilW C18 column (250 mm  4.6 mm i.d., 10 mm particle
size, 120 Å pore size) was used and the detector was set at 210 nm. Statistical comparison of the results of the two methods
showed clearly that there was no significant difference between them. Copyright © 2012 John Wiley & Sons, Ltd.

Keywords: calcitonin; fluorescamine; spectrofluorimetry; derivatization

Introduction
Salmon calcitonin (sCT) is a polypeptide hormone comprised of
32 amino acids and the major physiological role of the sCT is to
control the concentration and metabolism of calcium in the
body (1). Owing to its ability to reduce osteoclast activity, sCT is
also commonly used in the treatment of bone diseases such as
Paget’s disease, hypercalcemia and osteoporosis. Because sCT is
degraded by intestinal enzymes and stomach acids when given
orally, it is administered via parenteral and nasal routes. Various
analytical methods have been described for the quantitative deter-
mination of sCT, including high-perforance liquid chromatography
(HPLC) with UV (2,3), fluorescence (4–6) and electrochemical
detection (7), liquid chromatography-electrospray ionization
mass spectrometry (LC-ESI-MS) (8,9), voltammetry (10),
fluoroimmunoassay (11) and spectrofluorimetry (12).
Peptides often lack a suitable chromophore, fluorophore or
electrophore and have to be detected by the UV absorption of
their peptide bonds within the wavelength range from 205 to
230 nm (13). This range, however, is very unspecific because
many substances show UV absorption therein. Only peptides
bearing at least one amino acid with an aromatic side chain
may be detected by their UV absorption in the range from 250
to 280 nm. Here, selectivity is sufficient but sensitivity is often
not high enough to detect low concentrations of peptides.
Fluorimetric derivatization procedures are promising in solving
sensitivity and selectivity problems. Comparing UV and fluores-
cence detection, it is obvious that the sensitivity of fluorescence is
some order of magnitude higher than that of UV detection (14).
Because some of the peptides bearing phenylalanine, tryptophan
or tyrosine show a weak fluorescence within the emission
wavelength range from 270 to 350 nm, they have to be derivatized
by introducing a fluorophore group. For that purpose, one possible
fluorophore is fluorescamine (FSM), a widely used derivatizing
agent for peptides bearing primary amino groups (15).
In this study, sCT in ampules was determined using a spectro-
fluorimetric method in which sCT was derivatized using FSM, a
fluorogenic derivatizing agent. FSM has several advantages over
other amine derivatizing reagents, including its commercial
availability in the pure form, ease of handling, high fluorescence
quantum yield and higher rate of reactivity with primary amines,
peptides and proteins (16). In addition, FSM and its hydrolysis
products are non-fluorescent and excess reagent is desroyed
in < 1 min (17). Thus, a simple, sensitive and selective spec-
trofluorimetric method has been developed for the determi-
nation of sCT in ampules. The results of spectrofluorimetric
measurements were compared with those obtained using
an isocratic HPLC method, as a reference. Compared with
other existing methods, e.g. HPLC, LC-ESI-MS and voltam-
metry, the proposed spectrofluorimetric method is simple,
selective and does not require expensive instrumentation
and a skilled operator.
* Correspondence to: Hasan Basan, Gazi University, Faculty of Pharmacy,
Department of Analytical Chemistry, 06330, etiler, Ankara, Turkey. Tel: +90-
3122023105; Fax: +90-3122235018. E-mail: basan@gazi.edu.tr
Gazi University, Faculty of Pharmacy, Department of Analytical Chemistry,
961

06330, etiler, Ankara, Turkey

Luminescence 2013; 28: 961–966 Copyright © 2012 John Wiley & Sons, Ltd.

Experimental
Apparatus
The derivatized sCT spectrum was obtained using a double-beam
UV–Vis spectrophotometer, Shimadzu UV-160A (Tokyo, Japon).
A Varian Carry Eclips spectrofluorimeter (Mulgrave, Victoria,
Australia) equipped with a 150 W xenon high-pressure lamp
was used throughout. Slits widths and excitation and emission
wavelengths were set to 10, 390 and 484 nm, respectively. pH
measurements were performed using an Orion Model 720 A with
a combined electrode (Beverly, MA, USA). Chromatographic
measurements were achieved using an Agilent 1100 model HPLC
system with a diode array detector and a reversed-phase NucleosilW
C18 (250 mm x 4.6 mm i.d., 10 mm particle size, 120 Å pore size)
column. The mobile phase consisted of a mixture of mobile phase
A and mobile phase B (60:40, v/v). Mobile phase A contained
900 mL of 0.060 M tetramethylammonium hydroxide at pH 2.5,
prepared by adding phosphoric acid, and 100 mL of acetonitrile.
Mobile phase B was obtained by mixing 400 mL of 0.040 M
tetramethylammonium hydroxide at pH 2.5, prepared by
adding phosphoric acid, and 600 mL of acetonitrile. The flow
rate, column temparature and injection volume were 1.0 mL/min,
65 C and 80 mL, respectively. Detector wavelength was set
at 210 nm.
Materials and reagents
sCT (molecular mass 3432 Da, 5253 IU/mg) was kindly donated
by Novartis (Ringaskiddy, Co. Cork, Ireland). MiacalcicW 100 IU
ampule (100 IU/mL) was purchased from Novartis Turkey. FSM,
FluramW, was obtained from Sigma (Steinheim, Germany).
All other chemicals were obtained from Merck (Darmstadt, Germany).
0.01 M acetate buffer, pH 3.4, was prepared by mixing appropriate
amounts of 0.01 M HCl and 0.01 M sodium acetate solutions.
Borate buffers, pH 6.5–9.5, were prepared by adding 5.0 M NaOH
to 0.5 M boric acid solutions. A stock solution containing 0.0124%
(w/v) FSM was freshly prepared in acetone. For the preparation
of a standard stock solution of sCT (1.0 mg/mL), 10 mg sCT was
accurately weighed and dissolved in acetate buffer, pH 3.4, and
the volume was then adjusted to 10 mL with acetate buffer. The
derivatization procedure was performed in a manner similar to
procedures described in our preliminary study (12). Aliquots
(5–60 mL) from 1.0 mg/mL stock sCT solution were transferred
into 10.0 mL volumetric flasks. To each flask, 5.0 mL of borate
buffer was added, followed by 0.75 mL of 0.0124% (w/v) FSM
solution. The flasks were shaken vigorously for 30 s and deriva-
tization was allowed to proceed for 3.5 min more after adjusting
the volume to 10.0 mL with distilled water. Standard solutions
of the derivatized sCT were in the range of 0.5–6.0 mg/mL.
Finally, emission spectra and fluorescence intensities of the
derivatized sCT solutions were measured by setting excitation
and emission monochromators and slit widths to 390, 484 and
10 nm, respectively.
During the HPLC study, sCT stock solution (1.0 mg/mL) was
prepared by dissolving 10 mg sCT in mobile phase A and the
volume was adjusted to 10 mL with mobile phase A. Appropriate
amounts from the stock solution were taken into 10 mL volu-
metric flasks in order to prepare standard solutions of sCT
in the range of 0.47–3.8 mg/mL. An 80 mL aliquot from each
standard was injected and the calibration curve was obtained by
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D. Koçdan and H. Basan
drawing peak area values corresponding to each standard versus
concentration of standars. By referring to the calibration graph,
the amount of sCT in ampules was successfully determined.
Analysis of ampules
The proposed spectrofluorimetric method was applied for the
determination of sCT in ampules. One milliliter of MiacalcicW
100 IU ampule was transferred into a 10 mL volumetric flask
and 5.0 mL of borate buffer was added. After adding 0.75 mL
of 0.0124% (w/v) FSM to this mixture, the flask was shaken for
30 s and derivatization was allowed to proceed for 3.5 min more
after adjusting the volume to 10.0 mL with distilled water. The
fluorescence signal was measured using a spectroflourimeter.
By referring to the calibration graph, the amount of sCT in
ampules was determined. sCT in ampules was also determined
using a modified isocratic HPLC as a reference method. One
milliliter of MiacalcicW 100 IU ampule was transferred into a
10.0 mL volumetric flask and volume was adjusted to 10.0 mL
with mobile phase A. Thereafter, 80 mL of this ampule solution
was injected into the HPLC system. The flow rate, column
temparature and injection volume were 1.0 mL/min, 65  C and
80 mL, respectively. By using the area corresponding to the sCT
chromatogram, the amount of sCT in ampules was determined
referring to the calibration graph.
Results and discussion
Specrofluorimetric method
Derivatization of sCT with fluorescamine. Because sCT contains
only one aromatic amino acid, tyrosine, and does not have sufficient
intrinsic fluorescence, determination of sCT is mostly performed
using HPLC with UV detection. In order to determine sCT using a
spectrofluorimetric method, fluorescamine, 4-phenylspiro[furan-2
(3H),1-phthalan]-3,3-dione, was selected as a derivatizing agent
because the product is highly fluorescent, whereas FSM and its
degradation or hydrolysis products are non-fluorescent,
resulting in a low background (17). Conventional amine-specific
fluorogenic reagents such as dansyl chloride and 7-chloride-4-
nitrobenzofurazan require heat and also react with water
producing potential interference (5). Another popular amine
specific fluorogenic reagent, O-phthaladehyde, is unsuitable
because it forms highly unstable derivatives with peptides
that also have low fluorescence yield. The most interesting point
in this derivatization reaction is that starting from two non-
fluorescent substances (namely sCT and FSM), a highly fluores-
cent product is generated. Another point which should be
emphasized is that because FSM is only soluble and stable in
some organic solvents, acetone was used as a solvent. Ethanol
was also tested but, interestingly, very low fluorescence was
obtained for the derivatization product of sCT and FSM. This
result is consistent with the conclusion of Castel et al. (18),
stating that alcohols interact in a reversible manner with FSM
leading to a slowing of the reaction with other nucleophiles
(e.g. sCT). The derivatization reaction of sCT with FSM is repre-
sented in Fig. 1. In this reaction, FSM reacted with the primary
amine group of sCT at the N-terminus. Excitation and emission
spectra of reaction product were plotted and are represented
in Fig. 2. The emission spectrum of the reaction product
confirms that sCT was successfully derivatized using FSM.
Luminescence 2013; 28: 961–966
A novel spectrofluorimetric method for the determination of calcitonin

Figure 1. Derivatization reaction between sCT and FSM.

Figure 3. Effect of pH on the fluorescence intensity of FSM-labeled sCT (2.0 mg/mL).

Figure 2. Excitation and emission spectra of FSM-labeled sCT (1.6 mg/mL).

Optimization of the reaction parameters

Derivatization parameters including borate buffer pH, borate
Figure 4. Effect of borate buffer volume on the fluorescence intensity of FSM-
buffer volume, FSM concentration and derivatization time were
labeled sCT (2.0 mg/mL).
carefully investigated and optimized.

Effect of pH. Reactions of amines with fluorescamine have

been shown to be pH dependent (19) and it was represented
that the fluorescence appeared only in alkaline medium and
completely disappeared in acidic medium. The reaction of FSM
with amino acids and peptides has a large workable pH range,
from pH 7.0 to pH 10.5 (20). For this reason, the effect of pH
on the fluorescence intensity of the derivatization product was
investigated at pH 6.5–9.5 (Fig. 3). The net fluorescence intensity
of the reaction product increased steadily and reached a
maximum value at pH 8.5. At much lower and upper pH levels,
diminished fluorescence intensity of sCT-FSM was observed.

Effect of borate buffer volume. During the optimization

study, borate buffer volumes in the range of 1.0–6.0 mL (0.5 M,
pH 8.5) were tested and the highest net fluorescence signal
and best correlation coefficients were obtained using 5.0 mL of
borate buffer (Fig. 4). The fluorescence intensity of the deriva-
tized sCT increased gradually and reached a maximum value at
5.0 mL. Beyond this volume, the fluorescence signal reduced
sharply. This tendency is consistent with the study conducted
by Zhu et al. (20). In their study, they showed that the fluores-
cence intensity decreased three times when the concentration
of borax at pH 10 was increased three times in the final reaction
solution. Thus, they concluded that the rate of fluorescamine
hydrolysis is increased at higher buffer concentrations.
Figure 5. Effect of FSM concentration in the reaction medium on the fluorescence
intensity of FSM-labeled sCT (2.0 mg/mL).
Effect of fluorescamine concentration. Figure 5 shows the ef-
fect of the concentration of FSM, in the reaction medium, on the
net fluorescence intensity of the product. It is obvious that the
highest fluorescence intensity value was obtained with a FSM
concentration of 9.3 mg/mL. Thereafter, fluorescence intensity
decreased sharply, probably due to the high blank signal, and
stayed almost constant above 12.4 mg/mL.
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 D. Koçdan and H. Basan

Effect of derivatization time. The influence of reaction time
on the fluorescence intensity of derivatized sCT was studied over
a time interval of 1–10 min. It is clearly evident from Fig. 6
that the highest fluorescence intensity was reached at 4 min.
Therefore, this was accepted as an optimum derivatization time.
Analytical performance
The validity of the proposed method was tested regarding
linearity, limit of detection, limit of quantification, accuracy,
precision and recovery.
Linearity. After the optimization procedure, a linear regression
equation was obtained (Table 1). It was clearly demonstrated
that there was a linear relationship between fluorescence inten-
sity and the concentration of sCT in the concentration range
0.5–6.0 mg/mL.
Limit of detection and quantification. Limits of detection
(LOD) and quantification (LOQ) were calculated using the relations
3.3 SD/m and 10 SD/m, respectively, where m is the slope of the
calibration curve and SD is the standard deviation of lowest
concentration of the calibration standard (n = 5). LOD and LOQ
values for the derivatized sCT were calculated to be 0.124 and
0.372 mg/mL, respectively (Table 1).
Accuracy and precision. In order to test the accuracy and
precision of the proposed method, intra- and interday (three
different days) studies were performed by spiking the sample
from the ampule with appropriate amounts of the stock solution
of sCT. The high recovery (99.0–103.0) and low %RSD (4.9–5.5)
values obtained confirmed the accuracy and precision of the
proposed spectrofluorimetric method (Table 2). The results of
the proposed method and reference HPLC method were statisti-
cally compared, using Student’s t-test and F-test (Fisher test),
and the experimental results are summarized in Table 3. It can
be concluded that there were no significant differences, at the
95% confidence level, between the proposed method and the
reference HPLC method regarding accuracy and the precision.

HPLC as a reference method

Figure 6. Effect of derivatization time on the fluorescence intensity of FSM-la- Quantitative determination of sCT in ampules was also performed
beled sCT (2.0 mg/mL). using HPLC. Figure 7(a) and (b) shows typical chromatograms
obtained using standard sCT solution and MiacalcicW ampules
containing 100 IU/mg sCT, respectively. During the chromato-
Table 1. Analytical parameters
graphic study, standard sCT and ampule solutions were isocrati-
Spectrofluorimetry HPLC cally eluted, forming well-shaped, symmetrical peaks, and well
separated from the solvent front. The HPLC method for the
Wavelength (nm) lex = 389; 210
lem = 484
Linearity range (mg/mL) 0.5–6.0 0.47–3.8 Table 3. Statistical comparison of the spectrofluorimetric
Correlation coefficient, R 0.9990 0.9994 method and HPLC
Equation of calibration curvea
Slope 120 65012 Labeled (IU) Found  RSD %
İntercept 24.7 -7858
Spectrofluorimetry 100 98.7  2.6
LOD (mg/mL) 0.124 0.040
HPLC 100 97.9  2.6
LOQ (mg/mL) 0.372 0.120
tcalculated = 0.15 ttheoretical = 2.31 p = 0.05
a
y = mx + n, where x is the concentration of sCT in mg/mL. fcalculated = 1.17 ftheoretical = 6.39 p = 0.05

Table 2. Intra- and interday precision and accuracy for the analysis of calcitonin in spiked ampule

Intraday (n = 3) Interday (n = 3)
a a
Added (IU) Recovery (%) RSD (%) Added (IU) Recovery (%) RSD (%)
30 102.0 5.4 30 103.0 4.9
Spectrofluorimetry 60 101.0 5.5 60 100.9 5.3
90 101.5 5.3 90 99.0 5.1
30 98.0 4.8 30 99.0 4.7
HPLC 60 97.5 4.5 60 98.2 4.4
90 97.0 4.7 90 97.0 4.9
a
1 IU of sCT is approximately equivalent to 0.19 mg sCT.
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A novel spectrofluorimetric method for the determination of calcitonin
Figure 7. Chromatograms representing standard sCT solution (a) and miacalcic ampule (b).
determination of sCT was a new modified form of the method
described in The European Pharmacopoeia (21) in that a gradient
elution is described. However, in this modified HPLC method, an
isocratic elution was achieved using a reversed-phase NucleosilW
C18 (250 mm  4.6 mm i.d., 10 mm particle size, 120 Å pore size)
column and mobile phase consisted of a mixture of mobile phase
A and mobile phase B (60:40, v/v). Thus, this newly developed HPLC
method does not need gradient elution, i.e. solvent programming,
and also it provides a relatively short retention time (~ 11 min) for
the elution of sCT. In the European Pharmacopoeia, the
corresponding retention time for sCT was given as 20 min (21).
Therefore, it is costly and time-consuming compared with the
modified method. LOD and LOQ values of 0.040 and 0.120 mg/mL
were obtained (Table 1), respectively. High recovery (97.0–99.0%)
(Table 2) and low RSD% (4.4–4.9) (Table 2) values confirmed the
accuracy and precision of the modified HPLC method. The
Student’s t-test and F-test results showed no significant difference,
at the 95% confidence level, between the results of the two
methods (Table 3).
Conclusion
The proposed spectrofluorimetric method has been successfully
applied for the determination of sCT in ampule formulations. It is
obvious that the proposed method is simple, rapid and low-cost
because the instrumentation it requires is simple and is not
expensive. In addition, it does not require any extraction procedure.
The proposed spectrofluorimetric method is also selective because
it uses both excitation and emission wavelengths during the
measurement. It can be easily concluded that the method is
suitable for the routine analysis of sCT in quality control laboratories.
The proposed method can also be easily applied for the
Luminescence 2013; 28: 961–966 Copyright © 2012 John Wiley & Sons, Ltd.
determination of sCT in other pharmaceutical formulations,
especially for the determination of sCT in nasal sprays, and
other pharmaceuticals containing primary amine groups.
Acknowledgement
We would like to thank to Gazi University, Scientific Research
Project Unit for their support to our study with a project number
02/2009-25. We also acknowledge Avantis Pharma and Novartis
companies for their kind sCT donation.
References
1. Torres-Lugo M, Peppas NA. Transmucosal delivery systems for
calcitonin: a review. Biomaterials 2000;21:1191–6.
2. Mayer WJ, Long DA, Parikh DK. Phosphate-gradient high perfor-
mance liquid chromatographic method for the analysis of synthetic
salmon calcitonin. J Chromatogr A 1991;536:131–5.
3. Lee IH, Pollack S, Hsu SH, Miksic JR. Influence of the mobile phase on
salmon calcitonin analysis by reversed-phase liquid chromatography.
J Chromatogr Sci 1991;29:136–40.
4. Fukuda T, Ishikawa K, Imai K. Sensitive determination of salmon calcitonin
by means of pre-column derivatization, HPLC and fluorometric
determination. Biomed Chromatogr 1995;9:52–5.
5. Lee KC, Yoon JY, Woo BH, Kim CK, DeLuca PP. Post-column fluorescence
HPLC for salmon calcitonin formulations. Int J Pharm 1995;114:215–20.
6. Windisch V, Karpenko C, Daruwala A. LC assay for salmon calcitonin
in aerosol formulations using fluorescence derivatization and size
exclusion chromatography. J Pharm Biomed Anal 1992;10:71–6.
7. Lee HS, Choi SJ, Lee HM. Determination of salmon calcitonin in formula-
tions by high-performance liquid chromatography with electrochemical
detection. Chromatographia 1999;50:701–4.
8. Song KH, An HM, Kim HJ, Ahn SH, Chung SJ, Shim CK. Simple liquid
chromatography–electrospray ionization mass spectrometry method
for the routine determination of salmon calcitonin in serum. J Chromatogr
B 2002;775:247–55.
965
wileyonlinelibrary.com/journal/luminescence

9. D’Hondt M, Van Dorpe S, Mehuys E, Deforce D, De Spiegeleer B.
Quality analysis of salmon calcitonin in a polymeric bioadhesive
pharmaceutical formulation: sample preparation optimization by
DOE. J Pharm Biomed Anal 2010;53:939–45.
10. Osteryoung JG, Wikiel KJ. Determination of salmon calcitonin by
square wave adsorptive stripping voltammetry. Anal Chim Acta
1997;351:65–71.
11. Rong HQ, Torring O, Saaf M. Sensitive time-resolved fluoroimmu-
noassay of salmon calcitonin. Clin Chem 1994;40:1774–7.
12. Basan H, Gümüşderelioğlu M, Orbey MT. Release characteristics of
salmon calcitonin from dextran hydrogels for colon-specific delivery.
Eur J Pharm Biopharm 2007;65:39–46.
13. Koller M, Eckert H. Derivatization of peptides for their determination
by chromatographic methods. Anal Chim Acta 1997;352:31–59.
14. Kai M, Kojima E, Ohkura Y, Iwasaki M. High-performance liquid
chromatography of N-terminal tryptophan-containing peptides with
precolumn fluorescence derivatization with glyoxal. J Chromatogr A
1993;653:235–40.
15. Böhlen P, Stein S, Stone J, Udenfriend S. Automatic monitoring of
primary amines in preparative column effluents with fluorescamine.
Anal Biochem 1975;67:438–45.
966
wileyonlinelibrary.com/journal/luminescence Copyright © 2012 John Wiley & Sons, Ltd.
D. Koçdan and H. Basan
16. Boppana VK, Miller-Stein C. High performance liquid chromatographic
determination of peptide drugs in biological fluids by means of
pre- and post-column fluorescence derivatization techniques. Anal
Chim Acta 1997;352:61–9.
17. Udenfriend S, Bohler S, Dairman W, Leimgruber W, Wiegele M.
Fluorescamine: a reagent for assay of amino acids, peptides,
proteins and primary amines in the picomole range. Science
1972;178:871–72.
18. Castel JV, Cervera M, Marco R. A convenient micromethod for
the assay of primary amines and proteins with fluorescamine. A
reexamination of the conditions of reaction. Anal Biochem
1979;99:379–91.
19. Walsh MI, Belal F, El-enany N, El-maghrabey MH. Simple and sensitive
spectrofluorimetric method fort he determination of pregabalin in
capsules through derivatization with fluorescamine. Luminescence
2011;26:342–8.
20. Zhu R, Kok WTh. Postcolumn derivatization of peptides with fluorescamine
in capillary electrophoresis. J Chromatogr A 1998;814:213–21.
21. The European pharmacopoeia, European Directorate for the Quality of
Medicines and Healthcare, Strassbourg, France, 5th edition. 01/2005:
monograph 0471, p 1148.
Luminescence 2013; 28: 961–966