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FARMACIA, 2008, Vol.LVI, 4
SPECTROPHOTOMETRIC METHODS FOR
QUANTITATIVE DETERMINATION OF SOME
WATER SOLUBLE RUTIN DERIVATIVES
ANA-MARIA DĂNILĂ
Department of Analytical Chemistry, Faculty of Pharmacy, University of
Medicine and Pharmacy “Gr.T.Popa”, Universitatii Street 16, Iasi,
700015, Romania
*
corresponding author: ana_maria22us@yahoo.com
Abstract
The aim of this study was to establish, by ultraviolet-visible absorbtion
spectroscopy some sensitive and selective methods for determination of some water soluble
rutin derivatives. We established 14 sensitive, selective and rapid methods for the
determination of seven water soluble rutin derivatives, two methods for each compound.
These methods can be successfully applied to assay water soluble rutin derivates in real
samples.
Rezumat
Scopul acestei lucrări este stabilirea, prin spectrofometrie în ultraviolet – vizibil,
unor metode sensibile şi selective pentru determinarea unor derivaţi hidrosolubili ai
rutozidului. Au fost elaborate 14 metode sensibile, selective şi rapide pentru determinarea a
şapte derivaţi hidrosolubili ai rutozidului, câte două metode pentru fiecare compus. Aceste
metode pot fi utilizate cu succes pentru determinarea derivaţilor hidrosolubili ai rutozidului
din probe reale.


water soluble rutin derivatives
spectrophotometry
INTRODUCTION
Flavonoids are polyphenolic compounds that exist ubiquitously in
vegetal food [1]. They show various interesting biological activities, such as
scavenging of oxygen radical, anti-cancer action, modulation of high blood
pressure, antibacterial, antibiotic and anti-allergy actions. So far, over 4000
structurally unique flavonoids have been isolated from vegetal sources [2].
It has been elaborated a spectrophotometric assay for the
determination of hidrosoluble rutin derivatives from aqueous solutions,
which were prepared at Pharmaceutical Chemistry Department, „Gr. T.
Popa” University of Medicine and Pharmacy, Iasi [3].
Figure 1 shows the water soluble rutin derivatives structure.
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FARMACIA, 2008, Vol.LVI, 4
HO
HO
OH
HO
CH2
CH2
N
N
CH2
CH
CH2
O
OH
N
H5C2
O
N
CH2
CH
OH
CH2
O
O
OH
O
HO
rham
glu
O
O
HO
I
rham
II
HO
HO
OH
OH
H5C6
N
N
CH2
CH
CH2
O
H2N
O
CH2
CH2
N
N
CH2
CH
CH2
O
O
OH
OH
O
O
HO
III
rham
glu
HO
O
rham
IV
HO
OH
OH
N
N
CH2
CH
CH2
O
H3C
O
N
N
NH
CH2
CH
CH2
O
HO
rham
O
O
glu
HO
O
V
VI
OH
rham
glu
O
HO
HO
OH
O
O
CH2
CH
CH2
N
N
OH
rham
O
OH
OH
glu
glu
O
HO
C2H5OOC
glu
O
CH2
CH
CH2
O
O
OH
O
O
O
OH
HO
rham
glu
O
VII
Figure 1
Structure of rutin hidrosoluble derivatives
I: 3-[[6 – 0 - (6 – deoxy - α-L - manopiranosil) - β - D -glucopiranosil] - oxi] - 2 - (3,4dihydroxiphenil) - 5 - hydroxi - 7 - (1-oxi-(β-hydroxi-propyl) - piperazin-4-etylhydroxi)-4H-1benzopirane-4-one; II: 3-[[6-0-(6-deoxy -  - L - manopiranosil) -  - D-glucopiranozil] -2(3,4 -dihydroxifenil)-5-hydroxi-7-(1-oxi-(β - hydroxi - propyl) - piperazin – 4-ethyl) - 4H - 1 benzopiran-4-one; III: 3-[[6-0-(6-deoxy--L-manopiranosil) -  - D- glucopiranozil] - oxi]-2(3,4-dihydroxiphenil)-5-hydroxi-7-(1-oxi - (- hydroxi - propyl) - piperazin
- 4 - phenil)-4H-1-benzopiran-4-one; IV: 3-[[6-0-(6-deoxy--L-manopiranosil)--D-gluc
opiranozil] - oxi] – 2-(3,4 -dihydroxiphenil) - 5 - hydroxi - 7 - (1-oxi - (-hydroxi-propil) piperazin - 4 -ethylamino) - 4H- 1 -benzopiran-4-ona; V: 3 - [[6 - 0 - (6-deoxy -  - L manopiranosil) -  - D -glucopiranozil] - oxi] - 2 - (3,4 - dihydroxiphenil) - 5-hydroxi - 7 - (1oxi-(-hydroxi-propyll)-piperazin-4-carboxiethyl)-4H-1 -benzopiran-4-one; VI: 3-[[6-0-(6deoxy--L-manopiranosil)--D - glucopiranozil] - oxi]-2- (3,4-dihydroxifenil)-5-hydroxi-7-(1oxi-(-hydroxi-propil) - amino – piperazin-4- methyl) - 4H -l-benzopiran-4-one; VII:1,4-Bis[3-[[6-0-(6-deoxy--L-manopiranosil)--D-glucopiranozil]-oxi]-2-(3,4-dihydroxiphenil)-5hydroxi-4H-1-benzopiran-4-on-7-(oxi - ( - hydroxi-propyl)] –piperazine
MATERIALS AND METHODS



water soluble rutin derivatives (>99%);
distilled water, purchased from Sigma-Aldrich;
UV-VIS Hewlet-Packard spectrophotometer, H.P. 8453
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Stock solutions between 12.5 ng/mL and 200 ng/mL of compounds
I – VI and 3.1 ng/mL and 100 ng/mL of compound VII had been prepared.
Absorbtion spectra were recorded between 200 and 800 nm using an UV-VIS
spectrofotometer, cuve 1 cm, with maxim at two wavelengths λ1 and λ2: λ1 =
258 nm and λ2 = 320 nm, for compound I, λ1 = 256 nm and λ2 = 343 nm, for
compound II, λ1=258 nm and λ2 = 348 nm, for compound III, λ1 = 256 nm
and λ2 = 348 nm, for compound IV, λ1= 259 nm and λ2 = 348 nm, for
compound V, λ1 = 256 nm and λ2 = 357 nm and for compound VI, λ1 = 256
nm and λ2 = 349 nm, for compound VII. Distiled water was used as a blank.
RESULTS AND DISCUSSION
Figure 2 shows absorbtion specra of the compounds which we
mentioned above. We present calibration curves for each compound in
figure 3 during the concentrations range mentioned above.
A
B
C
D
E
F
G
Figure 2
UV Spectra of compound I-VII (figure 2A-2G) at different concentrations:
50ng/mL, 100 ng/mL and 200 ng/mL
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In order to validate the elaborated method, we present linearity,
precision, detection limit and quantification limit.
1
1
0,9
0,9
0,8
0,8
0,8
0,7
0,6
0,5
0,4
0,3
0,1
0
0
258 nm
320nm
Linear (258 nm)
Linear (320nm)
300
0,6
0,5
0,4
0,3
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
0
B
100
200
300
0
0
C
Concentration (ng/mL)
1
1
0,9
0,9
0,8
0,8
0,8
0,5
0,4
0,3
0,7
Absorbanţa (AU)
0,6
0,6
0,5
0,4
0,3
0,2
0,2
0
0
100
200
Concentration (ng/mL)
256nm
348nm
Linear (256nm)
Linear (348nm)
300
100
200
Concentration (ng/mL)
1
0,7
258nm
348 nm
Linear (258nm)
Linear (348 nm)
300
0,1
256nm
343nm
Linear (256nm)
Linear (343nm)
0,9
Absorbance (AU)
Absorbance (AU)
100
200
Concentration (ng/mL)
0,1
D
0,7
0,2
0,2
A
Absorbance (AU)
1
0,9
Absorbance (AU)
Absorbance (AU)
LINEARITY
To determine linearity, we used the average of 5 determination
series of concentrations between 12.5 ng/mL and 200 ng/mL. Compounds IVII absorbances determined at 2 wavelengths for each compound are listed
in table I. Calibration curves for these compounds performed at the above
range are presented in figure 3.
0,7
0,6
0,5
0,4
0,3
0,2
0,1
259nm
348nm
Linear (259nm)
Linear
300 (348nm)
0
0
E
100
200
0,1
0
F
Concentration (ng/mL)
0
100
200
300
256nm
357nm
Linear (256nm)
Linear (357nm)
Concentraţia (ng/mL)
1
0,9
Absorbanţa (AU)
0,8
0,7
0,6
0,5
0,4
0,3
0,2
256nm
349nm
Linear (256nm)
Linear (349nm)
0,1
0
G
0
100
200
300
Concentraţia (ng/mL)
Figure 3
Calibration curves for rutin soluble compounds: compound I (A); compound II (B);
compound III (C); compound IV (D); compound V (E), compound VI (F) and
compound VII (G)
For the compound I (figure 3A), the slope straight is 0.0042 (λ1)
and 0.0029 (λ2), respectively; the correlation coefficient is 0.9990 (λ1) and
0.9991 (λ2), respectively. For the compound II (figure 3B) the slope straight
is 0.0047 (λ1) and 0.003 (λ2), respectively; the correlation coefficient is
0.9997 (λ1) and 0.9998 (λ2), respectively. For the compound III (figure 3C)
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the slope straight is 0.0048 (λ1) and 0.0029 (λ2), respectively; the correlation
coefficient is 0.9988 (λ1) and 0.9998 (λ2), respectively. For the compound
IV (figure 3D) the slope straight is 0.0037 (λ1) and 0.0026 (λ2); the
correlation coefficient is 0.9996 (λ1) and 0.9998 (λ2), respectively. For the
compound V (figure 3E) the slope straight is 0.0048 (λ1) and 0.0034 (λ2);
the correlation coefficient is 0.9999 (λ1) and 0.9999 (λ2), respectively. For
the compound VI (figure 3F) the slope straight is 0.0035 (λ1) and 0.0026
(λ2); the correlation coefficient is 0.9997 (λ1) and 0.9993 (λ2), respectively.
For the compound VII (figure 3G) the slope straight is 0.0069 (λ1) and
0.0050 (λ2); the correlation coefficient is 1 for each wavelength.
For linearity determination we used the average values of 5
determinations series for concentration range between 12.5 ng/mL and 200
ng/mL. The calibration curves of compounds I – VII performed during
concentration range mentionated above are presented in figure 3. Spectra
were found to be linear with respect to the concentrations mentionated above.
PRECISION
In order to evaluate precision, it has been performed 5
determinations series of compounds I – VII, in different days. For all
compounds, the confidence interval was P = 95%; t = 2.78; n = 5. Tabel I
presents calculated parameters for each compound.
Compound
I
II
III
IV
V
VI
VII
Table I
Determination of recovery, standard deviation
and variation coefficient for compounds I – VII
Calculated parameters
Recovery
Standard deviation Variation coefficient
(%)
(SD) (%)
(CV) (%)
λ1
λ2
λ1
λ2
λ1
λ2
102.24
100.00
1.8466
2.7880
1.7997
2.7880
99.17
98.39
1.7097
1.8031
1.7240
1.8326
100.08
99.47
2.3758
1.9314
2.3739
1.9416
100.07
99.96
1.0416
2.0686
1.0408
2.0694
99.18
99.74
1.7556
1.9255
1.7701
1.9305
100.01
101.18
2.6552
2.4482
2.6549
2.4196
99.28
98.21
2.5450
1.9165
2.5634
1.9514
ACCURACY
For accuracy evaluation we calculated the relative error (Xd) for the
concentration range between 25 and 100 ng/mL for all the studied
compounds. The average of the relative error was less than 5% for each
compound. Table II presents the average of the relative error for each
compound.
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Table II
Determination of relative error for the compounds I – VII
Mean value of the relative error
Compound
(Xd) (%)
λ1
λ2
I
2.4500
2.1862
II
1.4266
1.8544
III
1.8522
1.4600
IV
1.2733
1.4566
V
1.6511
1.4788
VI
1.9900
1.9100
VII
1.8922
1.8811
DETECTION LIMIT
The detection limit (LD) is the lowest concentration level that can
be determined to be statistically different from a blank [4, 5].
In table III we present the detection limit for each compound.
Compound
I
II
III
IV
V
VI
VII
Table III
Detection limits of compounds I – VII
Detection limit (LD) (ng/mL)
λ1
λ2
7.92
7.55
3.89
3.60
8.43
3.72
4.94
3.23
2.87
2.38
4.45
6.34
0.60
0.66
QUANTIFICATION LIMIT
The limit of quantification (LQ) is the level above which
quantitative results may be obtained with a specified degree of confidence
[4, 5].
In table IV, we presented the quantification limit of each
compound.
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Compound
I
II
III
IV
V
VI
VII
Table IV
Quantification limit of compounds I – VII
Quantification limit (LQ) (ng/mL)
λ1
λ2
26.42
25.17
12.97
12.00
28.12
12.41
16.48
10.76
9.58
7.94
14.85
21.15
2.02
2.20
CONCLUSIONS
There have been elaborated 14 analytical methods for
spectrophotometric determination of compounds I – VII, two methods for
each compound. The elaborated methods were validated in concordance
with the validation parameters and they are sensitive, selective and fast.
Thus, using a small sample amount, this method may be an important
contribution to quality control in the real samples.
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