Stability-Indicating LC Method for Assayof Cholecalciferol

Namita D. Desai, Pirthipal P. Singh, Purnima D. Amin&, Satishkumar P. Jain

Pharmaceutical Sciences and Technology Division, University Institute of Chemical Technology (Autonomous), University of Mumbai,Matunga, Mumbai 400019, India; E-Mail: namitadd@rediffmail.com

Received: 22 July 2008 / Revised: 27 September 2008 / Accepted: 23 October 2008Online publication: 11 December 2008

Abstract

This paper discusses the development of a stability-indicating reversed-phase LC method foranalysis of cholecalciferol as the bulk drug and in formulations. The mobile phase wasacetonitrile–methanol–water 50:50:2 (v/v). The calibration plot for the drug was linear in therange 0.4–10 lg mL-1. The method was accurate and precise with limits of detection andquantitation of 64 and 215 ng, respectively. Mean recovery was 100.71%. The method wasused for analysis of cholecalciferol in pharmaceutical formulations in the presence of itsdegradation products and commonly used excipients.

Keywords

Column liquid chromatographyCholecalciferolStability-indicating

Introduction

Cholecalciferol, also known as vitamin

D3, is the molecule responsible for

calcium absorption for proper bone

development and muscle contraction

[1–3]. The literature reports a variety of

chemical, biological, and chromato-

graphic methods for analysis of vitamin

D3 [4, 5]. The LC method developed in

the work discussed in this paper is

advantageous because it enables stability-

indicating, accurate, specific, rapid,

and reproducible analysis of cholecal-

ciferol.

Experimental

Cholecalciferol (99.67% purity) was

obtained as a gift from Bajaj Healthcare,

Mumbai, India. Commercial tablets and

suspension obtained locally, and Meltlets

produced in-house were analyzed by use

of the proposed method. The components

of placebo formulations were calcium

citrate, Avicel, mannitol, Indion 414, so-

dium lauryl sulphate, methyl paraben,

and propyl paraben.

Liquid chromatography was per-

formed with a Jasco (Tokyo, Japan)

PU-980 intelligent pump coupled with a

Jasco MD-2015 (plus) multiwavelength

detector and a model 7725 Rheodyne

injector with 20 lL sample loop. Data

processing was performed with Borwin

Chromatography software version 1.50

for LC peak integration. Compounds

were separated on a 4.6 mm 9 250 mm

i.d., 5 lm particle, Waters Spherisorb

RP-18 ODS2 column with acetonitrile–

methanol–water 50:50:2 (v/v) as mobile

phase at a flow rate of 1.2 mL min-1.

Before use the mobile phase was filtered

through a 0.45 lm pore size Ultipor N66

Nylon membrane (Pall, Mumbai, India)

and degassed by sonication. Chroma-

tography was performed at room tem-

perature under isocratic conditions.

Detection was at 254 nm and detector

sensitivity was set at 0.16 AUFS.

A standard solution (5 lg mL-1) of

cholecalciferol was prepared in themobile

phase. Samples of solid oral dosage forms

containing approximately 1,000 IU

cholecalciferol were crushed to a fine

powder whereas liquid samples contain-

ing approximately 500 IU cholecalciferol

2009, 69, 385–388

DOI: 10.1365/s10337-008-0914-x0009-5893/09/02 � 2008 Vieweg+Teubner | GWV Fachverlage GmbH

Limited Short Communication Chromatographia 2009, 69, February (No. 3/4) 385

were analyzed without pretreatment. The

samples, in duplicate, were quantitatively

transferred to amber volumetric flasks

(capacity 25 mL) and stirred with mobile

phase for 2 h. The solutions were centri-

fuged for 5 min at 3,000 rpm by use of a

Superfit (Mumbai, India) centrifuge then

injected for chromatographic analysis.

Intentional degradation (n = 3) was

attempted by use of heat, light, acid, base,

and oxidizing agent. Thermal degrada-

tion was attempted by heating 0.5 mL

100 lg mL-1 cholecalciferol at 80 �C for

60 min. Degradation with acid was

attemptedbyheating0.5 mL100 lg mL-1

cholecalciferol with 0.5 mL 0.1 M

hydrochloric acid at 80 �C for 15 min.

Degradation with base was attempted by

heating 0.5 mL 100 lg mL-1 cholecal-

ciferol with 0.5 mL 0.1 M sodium

hydroxide at 80 �C for 15 min. Photo-

degradation was attempted by exposing

0.5 mL 100 lg mL-1 cholecalciferol to

sunlight for 60 min. Oxidative degrada-

tion was attempted by heating 0.5 mL

100 lg mL-1 cholecalciferol with 0.5 mL

hydrogen peroxide (30%) for 15 min. All

these solutions except that for photodeg-

radation were prepared in amber volu-

metric flasks. After completion of the

degradation treatments the samples were

cooled to room temperature, neutralized

(where required), diluted to 10 mL with

the mobile phase, and injected for chro-

matographic analysis. The degraded

samples were analyzed by comparison

CholecalciferolRt = 6.6 min

(d)

ThermalDegradation product

(Rt = 5.4 min)

Acid degradation product(Rt = 1.8 min, 6.1 min)

(b)

(f)

PhotoDegradation product

(Rt = 5.4 min)

Base degradationproduct

(Rt = 1.6 min)

OxidativeDegradation

product(Rt = 5.9 min)

(e)

(c)

(a)

Time (min) Time (min)

Fig. 1. Chromatograms obtained from cholecalciferol and its degradation products: a standard, 10 lg mL-1; b acidic degradation (63.78%);c basic degradation (83.50%); d oxidative degradation (53.74%); e photodegradation (13.44%); f thermal degradation (11.82%)

386 Chromatographia 2009, 69, February (No. 3/4) Limited Short Communication

with a control sample (lacking degrada-

tion treatment).

The method was validated in accor-

dance with recognized guidelines [6–9].

To demonstrate the specificity of the

method, placebo formulations containing

the excipients but no drug were subjected

to themethods of sample preparation and

analysis described above. The stability of

cholecalciferol in the mobile phase was

assessed by injecting a standard solution

(5 lg mL-1) 0, 8, and 24 h after prepa-

ration (n = 3). System precision was

evaluated by performing triplicate analy-

sis of cholecalciferol at three concentra-

tions (1, 3, and 5 lg mL-1). Method

precision was determined by analysis of

cholecalciferol standards at three differ-

ent concentrations (1, 3, and 5 lg mL-1).

The accuracy of the method was deter-

mined by measurement (n = 3) of recov-

ery using pellets from the same lot of the

developed formulation containing 50,

100, and 150%. LOD and LOQ were

determined as the amounts for which the

signal-to-noise ratios were 3:1 and 10:1,

respectively. Eight solutions containing

0.4–10 lg mL-1 cholecalciferol were

prepared in mobile phase. Peak area and

concentration data were treated by least-

squares linear regression analysis (n = 3).

Results and Discussion

The retention time of cholecalciferol

under the chromatographic conditions

described above was 6.6 min (Fig. 1a).

Chromatograms obtained from the

degraded samples are as shown in

Fig. 1b–f. The peaks of the degradation

products were well resolved from that of

cholecalciferol (RS > 3). A single peak

at 6.68 min was observed in chromato-

grams of the drug samples extracted

from the marketed formulations and

from pellets developed in-house; there

was no interference from the excipients

commonly present in the formulations.

It may therefore be inferred that no

degradation of cholecalciferol in the

pharmaceutical formulations was

detected by use of this method (Table 1).

In validation of the assay, placebo

formulation samples yielded clean chro-

matograms with no interference from the

excipients; this is indicative of the speci-

ficity of the method. Cholecalciferol was

found to be stable in the mobile phase,

because no peaks corresponding to deg-

radation products were observed and

there was no significant change in the

peak area of the drug (RSD <1%)

(Table 1). The method was found to be

precise (RSD 0.873%) and accurate

(RSD 1.07%) (Table 1). The recovery

data listed in Table 1, obtained from a

study of the pellet formulation, ranged

from 99 to 102% with low RSD (1.08%).

This quantitative recovery of cholecal-

ciferol indicates there was no interfer-

ence from excipients present in the

formulation. The LOD and LOQ were

64 and 215 ng, respectively. A plot of

drug peak area against concentration

was linear over the concentration range

0.4–10 lg mL-1. The regression equa-

tion calculated by the least-squares

method was y = 38,285x + 827.8,

correlation coefficient 0.9999.

Conclusion

This RP-LC method for assay of

cholecalciferol is precise, specific, rapid,

and stability-indicating. The method

may be used to assess the stability of

cholecalciferol as the bulk drug and in

its pharmaceutical formulations. Chro-

matographic analysis time of less than

10 min was advantageous for use of the

method in routine analysis. It may be

extended to study of the degradation

kinetics of cholecalciferol and also for

Table 1. Results from validation of the method

Cholecalciferol concentration Peak areas RSD (%)

Stability in the mobile phase5 lg mL-1 199,948 199,653 199,698 0.115 lg mL-1 199,422 199,863 199,8755 lg mL-1 199,968 199,662 199,421

System precision1 lg mL-1 38,072 38,421 38,781 0.8733 lg mL-1 117,454 116,089 11,54075 lg mL-1 193,307 193,712 196,765

Method precision1 lg mL-1 38,072 37,775 37,832 1.073 lg mL-1 117,454 119,421 114,0705 lg mL-1 193,307 194,119 195,505

Recovery studiesConcentration Recovery (%) Average recovery (%)1 lg mL-1 (50%) 101.79 100.23 100.65 100.712 lg mL-1 (100%) 99.91 101.32 99.823 lg mL-1 (150%) 101.84 100.65 100.25

Analysis of formulationsCholecalciferol content ± SD (%)Marketed formulation (tablets) Marketed formulation

(suspension)In-house-developed

formulation (pellets)148.68 + 4.89 130.19 + 9.72 159.82 + 2.11

Limited Short Communication Chromatographia 2009, 69, February (No. 3/4) 387

analysis of the drug in plasma and other

biological fluids.

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