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Novel Approach for the Determination of

Alfacalcidol in Bulk and Tablet Dosage Form
using Spectrophotometric...

Article in LATIN AMERICAN JOURNAL OF PHARMACY · May 2013

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 Latin American Journal of Pharmacy Short communication
(formerly Acta Farmacéutica Bonaerense) Received: September 19, 2013
Revised version: April 27, 2013
Lat. Am. J. Pharm. 32 (5): 784-8 (2013) Accepted: May 1, 2013

Novel Approach for the Determination of Alfacalcidol in Bulk

and Tablet Dosage Form using Spectrophotometric Method
Syed SAEED-UL-HASSAN 1, Imran TARIQ 1*, Sabiha KARIM 1,
Muhammad KHALIL-UR-REHMAN 1 Sajid BASHIR 2 &, Ghulam MURTAZA 3

University College of Pharmacy, University of the Punjab, Lahore -5400, Pakistan.

1
2 Department of Pharmacy, University of the Sargodha, Sargodha
3 Department of Pharmaceutics, COMSATS Institute of Information Technology, Abbottabad, Pakistan

SUMMARY. The purpose of the study was to develop a new simple, accurate, precise and economic spec-
trophotometric method in UV-VIS region for the determination of alfacalcidol in bulk and pharmaceutical
tablet dosage form. The proposed method was validated in terms of linearity, accuracy, precision, limits of
detection and limits of quantification. It was found that Beer’s law was obeyed in range of 0.1-10 μg/mL
for alfacalcidol having correlation coefficient (R2) of 0.999. The method has been successfully applied for
the analysis of drugs in tablet formulation as well. The percent recovery was found from 99.69 to 100.11%
which indicate that the method is precise and reproducible. Limit of detection (LOD) and Limit of quan-
tification (LOQ) of drug were 0.09623 and 0.29161 μg/mL, respectively. Interday and intraday studies
showed good repeatability of the applied method. Percentage relative standard deviation values were
found less than 2% for proposed methods which illustrate the good precision. It was concluded that pro-
posed method was versatile, cheaper, accurate, selective, precise and rapid for the analysis of alfacalcidol.

INTRODUCTION ture 9 and in the treatment of secondary hyper-

The scope of developing and validating ana- parathyroidism 10. Different analytical methods
lytical method is to establish a suitable analytical have been reported for the determination of al-
method for a particular active ingredient in a facalcidol in bone mass which include liquid
more specific, accurate and precise way to im- chromatography 11, mass spectormetery 12, high
prove the condition and parameter, which performance liquid chromatography and Fourier
should be followed in the development and val- transform infra-red spectroscopy 13, but there is
idation 1. Alfacalcidol has been used as a vita- no method available which should be easy and
min supplement due to it longer half-life 2. It is
an active metabolite of Vitamin D, which per-
forms important functions in regulation of the
calcium balance and the bone metabolism 3. Al-
facalcidol has many chemical names i.e. 1-hy-
droxycholecalciferol, 1alpha-hydroxycholecalcif-
erol, 1 alpha-hydroxyvitamin D3 [(1R, 3S, 5Z)-5-
{}-4-methylidenecyclohexane-1, 3-diol] 4 (Fig 1).
Early administration of alfacalcidol can safely
and beneficially alter the natural course of renal
bone disease in patients with mild to moderate
renal failure 5. It has superior effects over plain
vitamin D plus calcium in glucocorticoid-in-
duced osteoporosis 6-8, prevention of hip frac- Figure 1. Chemical structure of alfacalcidol.

KEY WORDS: Alfacalcidol, Method validation, UV-Visible spectrophotometry.

* Author to whom correspondence should be addressed. E-mail: imran_1982@hotmail.com

784 ISSN 0326-2383

 Latin American Journal of Pharmacy - 32 (5) - 2013

less time consumable so there was a need of

analysis of alfacalcidol by simple UV spec-
trophotometer. No literature was also found for
the analysis of alfacalcidol as drug in bulk and
pharmaceutical dosage form by UV spectropho-
tometric method. The present study was aimed

Absorbance
to develop a new spectrophotometric method
which should be simple, accurate, precise and
reproducible for determination of alfacalcidol.

MATERIAL AND METHODS

Materials
Standard sample of alfacalcidol (purity
99.99%, Jawa pharmaceuticals (Pvt) Ltd. Lahore),
methanol (Merck Ltd., Germany), and alfacare
Wavelength (nm)
tablets (purity 99.71%, Cherwell pharma, Lahore,
Pakistan). Figure 2. λ max of alfacalcidol by UV scanning.

Instruments
Double beam Spectrophotometer UV-2550,
Shimadzu, Japan. Analytical balance Metler Absorbance

Toledo (Pvt) Ltd, Sonicator wtw, Germany.

Preparation of standard stock solution

Stock solution of alfacalcidol (10 µg/mL) was
prepared by dissolving 1 mg of standard drug in
50 mL of methanol using 100 mL volumetric
flask. Shake and sonicate the solution for 20
Concentration (μg/mL)
min and volume was made upto the mark with
methanol to get the concentration of 10 µg/mL. Figure 3. Linearity determination of alfacalcidol.
Filter the resultant stock solution using What-
mann filter paper (no. 41) to get the clear solu-
tion. No. Concentration Absorbance
(µg/mL)
Determination of λ max
1 0.1 0.001
The stock solution (10 µg/mL) was scanned
2 0.2 0.008
between 200-400 nm wave length to found λ
3 0.3 0.011
max of drug using methanol as blank. Alfacalci-
4 0.4 0.019
dol showed absorption maxima at 264 nm (Fig.
5 0.5 0.023
2).
6 0.6 0.04
7 0.7 0.051
Preparation of Calibration curve
8 0.8 0.059
From the stock solution of alfacalcidol (10
9 0.9 0.065
µg/mL) various concentrations of drug from 0.1-
10 1 0.073
10 µg/mL were prepared by transferring aliquots
11 2 0.131
of stock solution to volumetric flasks of 10 mL
12 3 0.201
and made upto the mark according to the con-
13 4 0.273
centration required. Their absorbances were
14 5 0.343
noted at their above selected respective λ max,
15 6 0.42
calibration curve were plotted as absorbance vs
16 7 0.494
concentration (Fig. 3) and their linearity range
17 8 0.563
was determined (Table 1).
18 9 0.634
19 10 0.704
Preparation of sample solution
Alfacalcidol (100 tablets, each containing 1 Table 1. Beer’s law range for linearity determination.

785
SAEED-UL-HASSAN S., TARIQ I., KARIM S., KHALIL-UR-REHMAN M., BASHIR S. & MURTAZA G.

µg of alfacalcidol) were triturated. From the trit- RESULTS

urate of hundered tablets quantity equivalent to Linearity
10 µg of drug was weighed, transferred to a 10 Nineteen concentrations of alfacalcidol
mL volumetric flask, sonicated with 5 mL of equivalent to 0.1-10 µg/mL were prepared and
methanol for 30 min, made up to volume with checked for their absorbance. The linearity of
solution and filtered with Whatmann filter paper detector response was established by plotting a
(no. 41) to get concentration of 1 µg/mL. From graph between concentration and absorbance of
this solution, suitable aliquots were prepared, alfacalcidol standard drug (Fig 3). The detector
scanned in UV region and absorbance was not- response was found to be linear for these dilu-
ed at 264 nm. tions and correlation coefficient (R2) value was
equal to 0.999 at wavelength 264 nm. The Y-
Statistical analysis equation for alfacalcidol concentrations was y =
The comparison of various parameters was 0.070x - 0.005 (Table 2).
conduced to elaborate the significant difference
using software, SPSS version 13.0. The level of Limit of Detection and Limit of
significance was set at 0.05. Quantification
LOD was found to be 0.09623 µg/mL while the value
Validation of the method of LOQ was 0.29161 µg/mL. the results of LOD and
The current method was validated according LOQ of drug showed sufficient sensitivity of the
to the international council of harmonization method (Table 3).
(ICH) guidance in terms of linearity, accuracy,
Parameters Alfacalcidol
precision, limits of detection, limits of quantifi-
cation and selectivity. Beer’s law range /µg ml–1) 0.1-10
Linearity Slope 0.070
Linearity of the proposed methods was de-
termined by analyzing the solutions of alfacalci- Intercept 0.005
dol in the range of 0.1-10 µg/mL. (Fig. 3) Correlation co-efficient 0.9999
LOD and LOQ Molar absorptivity 28202
The limit of detection (LOD) and limit of y- equation 0.070x - 0.005
quantification (LOQ) was then established by
evaluating the minimum level at which drug so- Table 2. Linearity parameters of alfacalcidol.
lution can be readily quantified with accuracy.
LOD and LOQ were calculated according to the
3.3 and 10 δ/s criteria, respectively, where δ is LOD (µg/mL) LOQ (µg/mL)
the standard deviation of the response and s is 0.09623 0.29161
the slope of the corresponding calibration
curve. Table 3. Limit of Detection and Limit of Quantifica-
Accuracy tion.
Accuracy of the method was determined by
performing recovery studies by standard addi- No. Interday Intraday
tion method in which pre analyzed samples precision % precision %
were taken and standard drug was spiked at 1 99.69 99.42
three different levels i.e. 80, 100, and 120%. The
2 99.43 99.25
% recovery and % RSD were calculated which
3 100.08 99.54
should be less than 2%.
Precision 4 99.23 99.01
System precision was estimated from re- 5 99.02 99.84
peatability and reproducibility. The intraday and 6 99.58 99.32
interday precision of the method were con- Mean 99.505 99.39
firmed by measuring absorbance of six replicate
S.D 0.3706 0.2809
samples of alfacalcidol three times in a day and
%RSD 0.372 0.282
also on two different days. Percentage relative
standard deviation (% RSD) values should less Table 4. Determination of Interday and Intraday Pre-
than 2%. cision.

786

Amount taken Amount added
Level
(µg/mL) (µg/mL)
80% 1.0 0.8
100% 1.0 1.0
120% 1.0 1.2
Table 5. Recovery of a sample solution of alfacalcidol added to sample and analysed by the proposed UV spec-
trophotometric method.
Precision
The reproducibility of the methods was
checked by measuring absorbance of six repli-
cates of 1 µg/mL of alfacalcidol intraday and in-
ter day basis. Percentage relative standard devia-
tion (% RSD) values were found less than 2%
that illustrates the good precision of the pro-
posed method (Table 4).
Accuracy
Accuracy of the procedure was determined
by comparing the analytical amount determined
vs. known amount spiked at 80, 100, and 120%
drug concentration. Percentage recovery of pro-
posed method was found 99.69 to 100.11% with
% RSD value was not more than 0.419 (Table 5).
DISCUSSION
The proposed method was applied to the
determination of alfacalcidol in bulk and in
commercial tablets. Various researchers have
studied the evaluation of alfacalcidol in bone
mass. However, the literature survey has shown
that no analytical study regarding spectrophoto-
metric method was available for its analysis. But
upon validation its parameters were also found
with in specified range. Beer’s law was obeyed
between 0.1 to 10 µg/mL, showed good correla-
tion coefficient (R2 = 0.999 value) with a mini-
mum detection limit (LOD) of 0.09623 µg/mL
and a quantification limit (LOQ) of 0.29161.
These values were found to be more accurate
than previously reported spectrophotometric
methods 14 of different analytes where LOD and
LOQ values are higher and show more variation
than the proposed method. Moreover, to check
the validity of the proposed method, dosage
forms were also tested for possible interference
with standard addition method. The differences
between the amount claimed and those mea-
sured were very low and results were within the
acceptable windows mentioned in the pharma-
copoeias. The mean percentage recoveries ob-
tained after six repeated experiments were with-
Latin American Journal of Pharmacy - 32 (5) - 2013
% Recovery
% R.S.D
(Mean ± %S.D)
99.69 ± 0.417 0.419
100.11 ± 0.110 0.110
99.84 ± 0.353 0.354
in B.P 2011 (95.0-105.0%) and U.S.P 2009 (90.0-
110.0%) specification for alfacalcidol having %
RSD values less than 2% indicating that results
are accurate and there is no interference from
the excipients. Similar results were reported by
Ayman & Alaa 15 and Ashok et al. 16. So the pro-
posed method was more reproducible with high
recoveries than the reference method, they can
be recommended for the routine analysis in the
majority of drug quality control laboratories.
CONCLUSION
It was concluded from the study that the
proposed method was simple, accurate and pre-
cise method for the analysis of alfacalcidol in
bulk and pharmaceutical tablet dosage form. All
the statistical parameters along with recovery
data confirm the reproducibility and accuracy of
methods. During analysis no interference with
the formulation excipients confirms the selectivi-
ty as well as specificity of proposed methods.
The methods were validated for various other
parameters as per ICH guidelines in which they
were found to be within the acceptance criteria.
Hence the methods can be satisfactorily applied
in quality control analysis of alfacalcidol.
REFERENCES
1. Nash, R.A. & Wachter, A. (2003) Pharmaceuti-
cal Process Validation; Published by Marcel
Dekker Inc., pp. 507-21.
2. Nuti, R., G. Bianchi, R. Nuti, G. Bianchi, M.L.
Brandi, R. Caudarella, E. D’Erasmo, C. Fiore et
al. (2006) Rheumatol. Intl. 26: 445-53.
3. PubChem Compound. 1-hydroxycholecalcifer-
ol - Compound Summary (CID 5282181)
Availale at <http://pubchem.ncbi.nlm.nih.
gov/summary/summary.cgi?cid=5282181&loc=
ec_rcs>.
4. Wikipedia. Alfacalcidol. Available at <http://
en.wikipedia.org/wiki/Alfacalcidol>.
5. Hamdy, N.A., J.A. Kanis, M.N. Beneton, C.B.
Brown, J.R. Juttmann, J.G. Jordans, et al.
(1995) BMJ 310: 358-63
787
 SAEED-UL-HASSAN S., TARIQ I., KARIM S., KHALIL-UR-REHMAN M., BASHIR S. & MURTAZA G.
6. Ringe, J., A. Dorst, H. Faber, K. Ibach & J.
Preuss. (2004) Rheumatol. Int. 24: 63-70.
7. Shiraishi, A., S.Higashi, H. Ohkawa, N. Ku-
bodera, T. Hirasawa, I. Ezawa, et al. (1999)
Calcified Tissue Int. 65: 311-6.
8. Reginster, J.Y., D. Kuntz, W. Verdickt, M.
Wouters, L. Guillevin, C.J. Menkès, et al.
(1999) Osteoporos Int. 9: 75-81.
9. Moe, S., L. D. Wazny & J. E. Martin (2008)
Can. J. Clin. Pharmacol. 15: 36-43.
10. Chapuy, M. C., M. E. Arlot, F. Duboeuf, J.
Brun, B. Crouzet, S. Arnaud, et al. (1992) New
Engl. J. Med. 327: 1637-42.
11. Flann, B. and B. Lodge (1986) J. Assoc. Off.
Anal. Chem. 69: 1026-30.
788
View publication stats
12. Maunsell, Z., D. J. Wright & S.J. Rainbow
(2005) Clinical Chem. 51: 1683-90.
13. British Pharmacopoeia (2009) Alfacalcidol.
Monographs: Medicinal and Pharmaceutical
Substances. Available at <http://db.yaozh.
com/foreign/BP2009/1-2/1-2_44.pdf>.
14. Murtaza, G., S.A.Khan, A. Shabbir, A. Mah-
mood, M.H.H.B.Asad, K. Farzana, et al. (2011)
Sci. Res. Essays 6: 417-21.
15. Ayman, A.G. & S.A. Alaa (2010) Arab. J.Chem.
3: 159-65.
16. Kumar, A., L. Kishore, A.Nair & N. Kaur (2011)
Der Pharma Chemica 3: 279-91.