Journal of Scientific & Industrial Research

368 J SCI IND RES VOL 69 MAY 2010

Vol. 69, May 2010, pp. 368-372

A new method for spectrophotometric determination of colchicoside

B Narayana* and N S Divya
Department of Post Graduate Studies and Research in Chemistry, Mangalore University, Mangalagangothri 574 199, India

Received 24 November 2009; revised & accepted 02 February 2010

A facile spectrophotometric method is proposed for determination of Colchicoside (COLSID) in bulk and in dosage forms
using ceric ammonium sulphate (CAS). Method involves addition of a known excess of CAS to COLSID and determination of
residual CAS with a fixed amount of either Azure B (AB), measuring absorbance at 633nm or Safranin O (SO), measuring
absorbance at 519 nm. Calibration graphs are linear over 25.00-50.00 µgml-1, 22.00-38.00 µgml-1 and apparent molar absorptiv-
ity is calculated to be 2.28 x 104 l mol-1 cm-1 and 2.77 x 104 l mol-1 cm-1 for CAS-AB and CAS-SO respectively. Method is
successfully applied to assay of COLSID in pharmaceutical formulations.

Keywords: Azure B (AB), Ceric ammonium sulphate (CAS), Colchicoside (COLSID), Safranin O (SO)

Introduction the mark with distilled water in a 100 ml standard flask.

Colchicoside (COLSID), an alkaloid, extracted from Azure B (s d fine chem Ltd., India, 90% dye content,
Gloriosa superba Linn, also known as “Meadow µ= 1.0 Χ10 5 lmol -1 cm -1) (0.01%) and Safranin O
Saffron”, is used for Gout and also as anticancer drug1. (s d fine chem. Ltd., India, 90% dye content, µ= 1.1Χ104
COLSID, a glucosylated colchicine, is less harmful to lmol-1 cm-1) (0.01%) were prepared with distilled water.
human than other colchicines2-4. Due to the action of
Standard Solution of COLSID
COLSID on spindle fibre formation during cell division, A stock standard solution (1000 µg ml–1 ) was
plant has been identified as a potential anticancerous prepared by dissolving weighed amount of pure COLSID
drug5. Immunoassay6, capillary electrophoresis7, HPLC8- in distilled water and diluting to a known volume; it
11
and spectrophotometry12 methods have been reported was later diluted appropriately with water to get working
for determination of COLSID. This study presents a concentrations for use in spectrophotometric methods
simple and sensitive method for spectrophotometric A and B, respectively.
determination of COLSID that is based on oxidation of
drug by ceric ammonium sulphate (CAS) and residual Method A
oxidant is determined by using either Azure B (AB) or Different aliquots (25.00-50.00 µg ml-1) of COLSID
Safranin O (SO). were transferred into a series of 10 ml calibrated flasks
using a micro burette. To each flask, CAS (1 ml) was
Experimental added, shaken well and kept for 10 min. Then added AB
Materials (1.5 ml) and diluted to mark with distilled water.
A UV-2550PC (Shimadzu, Japan) visible Absorbance of each solution was measured at 633nm
spectrophotometer with matched 1cm quartz cells were (Fig. 1a).
used for all measurements. All chemicals used were of
analytical grade and all solutions were prepared in Method B
distilled water. A solution of CAS (0.2%) was prepared Different aliquots (22.00-38.00 µg ml-1) of COLSID
by dissolving CAS (0.2 g) in 2M H2SO4 and made up to were transferred into a series of 10 ml calibrated flasks
using a micro burette. To each flask, CAS (1 ml) was
*Author for correspondence added, shaken well and kept for 10 min. Then added SO
E-mail: nbadiadka@yahoo.co.uk (3.0 ml) and diluted to mark with distilled water.
 NARAYANA & DIVYA: A NEW METHOD FOR SPECTROPHOTOMETRIC DETERMINATION OF COLCHICOSIDE 369

2.5

Absorbance
1.5

0.5

0
0 10 20 30 40 50 60
Concentration, µg/ml

2.5

2
Absorbance

1.5

0.5

0
0 5 10 15 20 25 30 35 40
T im e ( m in )
Concentration, µg/ml

2.3
2.1 a
1.9
Absorbance

1.7
1.5
1.3
1.1
b

0.9
0.7
0.5
0 5 10 15 20 25 30 35
)
Time, min

Fig.1—Calibration curve for: a) COSID-Azure B system; b) COLSID- Safranin O system; c) A graph of change in absorbance with time
(a, COLSID- Azure B system; b, COLSID- Safranin O system)

Absorbance of each solution was measured at 519 nm up to volume with distilled water. Above solution was
(Fig. 1b). analyzed according the proposed procedures.

Analysis of Tablet Results and Discussion

A total of 10 tablets of Goutnil (Inga Method A and B involve oxidation of drug by CAS
Pharmaceuticals Ltd., India) were ground into fine and residual oxidant is determined by using AB and SO.
powder, dissolved in distilled water by stirring for 10 When known amount of CAS is added to drug solution,
min, transferred into a 100 ml standard flask and made drug gets oxidized; excess oxidant bleaches dyes to leuco
370 J SCI IND RES VOL 69 MAY 2010

Scheme 1

form (Scheme 1). Change in absorbance with time is Analytical Data

studied and it is found that after 10 min there is no such A linear correlation was found between absorbance
variation in absorbance of reaction mixtures (Fig. 1c). and concentration of drug. Optical parameters (molar
Reaction mixtures showed maximum absorbance at 633 absorptivity, Beer’s law limit values) were calculated
nm (Fig. 2) for Method A and at 519 nm (Fig. 3) for (Table 1). Limit of detection (LOD) and limit of
Method B. quantitation (LOQ) were calculated according to ICH
 NARAYANA & DIVYA: A NEW METHOD FOR SPECTROPHOTOMETRIC DETERMINATION OF COLCHICOSIDE 371

2.5 c

b
Absorbance

1.5

1
a

0.5

0
400 450 500 5 50 600 650 700 750

Wavelength, nm
Fig. 2—Absorption spectra for: a) COLSID + CAS + Azure B; b) COLSID + Azure B; c) Azure B

1 .4

c
1 .2

1
b
Absorbance
a b s o rb a n c e

0 .8

0 .6
a

0 .4

0 .2

0
0 1 00 2 00 3 00 4 00 5 00 6 00 7 00

Wavelength, nm
Fig. 3—Absorption spectra for: a) COLSID + CAS + safranin O; b) COLSID + safranin O; c) safranin O

guidelines13 as LOD =3.3 × σ/S and LOQ = 10 × σ/S, Analytical results are considered to be satisfactory for
where σ is standard deviation of y-intercept of regression level of concentrations examined (Table 2).
lines (standard deviation of response) and S is slope of
calibration curve. Linearity was evaluated by calculation Interference Study
of regression. Sensitivity of proposed methods is To investigate effect of excipients and fillers on
determined by calculating sandell’s sensitivity (µg/cm2/ measurements, standard addition method was carried
0.001 Abs unit), which can be defined as smallest weight out. It was observed that talc (70 mg), starch (40 mg),
of substance that can be detected in column of unit cross glucose (65 mg) and lactose (55 mg) did not interfere in
section. measurements.

Accuracy and Precision Application

For accuracy and precision, solutions containing Proposed method had been successfully applied for
different concentrations of drug were analyzed. determination of COLSID in dietary supplements.
372 J SCI IND RES VOL 69 MAY 2010

Table 1—Analytical parameters Table 2—Evaluation of accuracy and precision

______________________________________________________________________________
Amount taken Amount found RE SD RSD
Method A Method B
µgml-1 µgml-1 % µgml-1 %
lmax, nm 633 519 Azure B*
Beer's law limit, µg ml-1 25.00-50.00 22.00-38.00 25.00 25.22 0.88 0.44 1.75
Molar absorptivity, 2.28×104 2.77×104 30.00 30.13 0.43 0.25 0.83
lmol-1cm-1 35.00 35.18 0.52 0.48 1.30
40.00 40.16 0.40 0.34 0.85
Detection limit** 0.0218 0.0074
45.00 45.40 0.88 0.51 1.14
Quantitation limit** 0.0663 0.0022
Sandell's sensitivity 0.024 0.0197 Safranin O**
Regression equation* Y=a+bX Y=a+bX 20.00 20.36 0.20 0.06 0.32
28.00 27.93 0.25 0.13 0.46
Slope (b) 0.3968 0.5076
32.00 32.04 0.12 0.11 0.36
Intercept (a) 0.0376 -0.0009 36.00 35.86 0.38 0.11 0.32
Correlation coefficient 0.9990 0.9971 38.00 37.94 0.16 0.09 0.24
*Y is absorbance and X is concentration in µgml-1; **Calculated *Mean value of 4 determinations, **Mean value of 3 determina-
using ICH-guidelines tions

Table 3—Assay of tablets

______________________________________________________________________________ 2 Kiselev V V & Yavich P A, Methods of isolating alkaloids of the
Labeled amount Amount* found Label claim±SD colchicine series, Chem Nat Compd, 26 (1990) 502-509.
mg mg % 3 Anne P & Phillippe G, Influence of chemical characteristics of
Method A 0.50 0.501 100.18±0.14 soil on mineral and alkaloid seed contents of Colchicum
autumnale, Environ Exper Bot, 54 (2005) 101-108.
t-test=1.039
4 Yoshida K & Hayashi T, Colchicoside in Colchicum autumnale
Method B 0.50 0.500 100.04±0.12 Bulbs, Agric Biol Chem, 52 (1988) 593-594.
t-test=0.349 5 Chitra R & Rajamani K, Per se performance and correlation for
*Mean value of 5 determinations, Calculated Student's t-value at yield and its quality characters in Glory Lily (Gloriosa superba
95% confidence level is 2.77 L.), Acad J Plant Sci, 2 (2009) 39-43.
_________________________________________________________________________ 6 Poulev A, Deus-Neumann B, Bombardelli E & Zenk M H, Im-
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Proposed method is simple, sensitive and possesses colchicine family by reversed- phase high- performance liquid
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investigated drugs in raw materials and in
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11 Baerheim S A & Verpoorte R, Chromatography of Alkaloids:
Acknowledgement Thin-layer chromatography (Elseveir Scientific Publishing
N S Divya thanks UGC-RFSMS scheme (under Company, New York) 1983, 470.
12 Stanislaus J, Smolensk, Frank A, Crane & Ralph F, Voigt chro-
SAP-Phase1) for providing financial help. matographic- spectrophotometric method for the separation and
determination of colchicines, J Am Pharm Assoc, 47 (2006)
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