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Stability-Indicating LC Method for Assay of Cholecalciferol

2009, 69, 385388

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:

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

This paper discusses the development of a stability-indicating reversed-phase LC method for analysis of cholecalciferol as the bulk drug and in formulations. The mobile phase was acetonitrilemethanolwater 50:50:2 (v/v). The calibration plot for the drug was linear in the range 0.410 lg mL-1. The method was accurate and precise with limits of detection and quantitation of 64 and 215 ng, respectively. Mean recovery was 100.71%. The method was used for analysis of cholecalciferol in pharmaceutical formulations in the presence of its degradation products and commonly used excipients.

Column liquid chromatography Cholecalciferol Stability-indicating

Cholecalciferol, also known as vitamin D3, is the molecule responsible for calcium absorption for proper bone development and muscle contraction [13]. The literature reports a variety of chemical, biological, and chromatographic 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, specic, rapid, and reproducible analysis of cholecalciferol.

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, sodium lauryl sulphate, methyl paraben, and propyl paraben. Liquid chromatography was performed 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 methanolwater 50:50:2 (v/v) as mobile phase at a ow rate of 1.2 mL min-1. Before use the mobile phase was ltered through a 0.45 lm pore size Ultipor N66 Nylon membrane (Pall, Mumbai, India) and degassed by sonication. Chromatography was performed at room temperature 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 the mobile phase. Samples of solid oral dosage forms containing approximately 1,000 IU cholecalciferol were crushed to a ne powder whereas liquid samples containing approximately 500 IU cholecalciferol

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

Cholecalciferol Rt = 6.6 min



Base degradation product (Rt = 1.6 min)

Oxidative Degradation product (Rt = 5.9 min)

Photo Degradation product (Rt = 5.4 min)

Thermal Degradation product (Rt = 5.4 min)


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%)

were analyzed without pretreatment. The samples, in duplicate, were quantitatively transferred to amber volumetric asks (capacity 25 mL) and stirred with mobile phase for 2 h. The solutions were centrifuged for 5 min at 3,000 rpm by use of a Supert (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 degradation was attempted by heating 0.5 mL

100 lg mL-1 cholecalciferol at 80 C for 60 min. Degradation with acid was attempted by heating 0.5 mL 100 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 cholecalciferol with 0.5 mL 0.1 M sodium hydroxide at 80 C for 15 min. Photodegradation 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 photodegradation were prepared in amber volumetric asks. 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 chromatographic analysis. The degraded samples were analyzed by comparison Limited Short Communication


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Table 1. Results from validation of the method

Cholecalciferol concentration Stability in the mobile phase 5 lg mL-1 5 lg mL-1 5 lg mL-1 System precision 1 lg mL-1 3 lg mL-1 5 lg mL-1 Method precision 1 lg mL-1 3 lg mL-1 5 lg mL-1 Recovery studies Concentration 1 lg mL-1 (50%) 2 lg mL-1 (100%) 3 lg mL-1 (150%) Analysis of formulations Cholecalciferol content SD (%) Marketed formulation (tablets) 148.68 + 4.89 Peak areas RSD (%)

199,948 199,422 199,968 38,072 117,454 193,307 38,072 117,454 193,307 Recovery (%) 101.79 99.91 101.84

199,653 199,863 199,662 38,421 116,089 193,712 37,775 119,421 194,119

199,698 199,875 199,421 38,781 11,5407 196,765 37,832 114,070 195,505




100.23 101.32 100.65

100.65 99.82 100.25

Average recovery (%) 100.71

Marketed formulation (suspension) 130.19 + 9.72

In-house-developed formulation (pellets) 159.82 + 2.11

with a control sample (lacking degradation treatment). The method was validated in accordance with recognized guidelines [69]. To demonstrate the specicity of the method, placebo formulations containing the excipients but no drug were subjected to the methods 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 preparation (n = 3). System precision was evaluated by performing triplicate analysis of cholecalciferol at three concentrations (1, 3, and 5 lg mL-1). Method precision was determined by analysis of cholecalciferol standards at three dierent concentrations (1, 3, and 5 lg mL-1). The accuracy of the method was determined by measurement (n = 3) of recovery 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 0.410 lg mL-1 cholecalciferol were prepared in mobile phase. Peak area and concentration data were treated by leastsquares 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. 1bf. 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 chromatograms 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 chromatograms with no interference from the excipients; this is indicative of the specicity of the method. Cholecalciferol was found to be stable in the mobile phase, because no peaks corresponding to degradation products were observed and there was no signicant 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 cholecalciferol indicates there was no interference 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 0.410 lg mL-1. The regression equation calculated by the least-squares method was y = 38,285x + 827.8, correlation coecient 0.9999.

This RP-LC method for assay of cholecalciferol is precise, specic, rapid, and stability-indicating. The method may be used to assess the stability of cholecalciferol as the bulk drug and in its pharmaceutical formulations. Chromatographic 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

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analysis of the drug in plasma and other biological uids.

1. DeLuca F (2004) Am J Clin Nutr 80:1689S1696S 2. Merck (1997) Manual of medical research information. Disorders of nutrition and metabolismsalt balance. Merck Research Laboratories, New Jersey, pp 671 673 3. Sweetman S (2002) Nutritional agents and vitaminsvitamin D. In: Martindalethe complete drug reference, 33rd edn. Pharmaceutical Press, London, pp 13911393

4. Koshy K, Beyer W (1984) Analytical prole of vitamin D3 (cholecalciferol). In: Florey K (ed) Analytical proles of drug substances, vol 13. Academic Press, Dublin, pp 656658 5. Krzek J, Moniczewska M, Zabierowska Slusarczyk G (1999) Acta Pol Pharm 56(4):261266 6. Krzek J, Moniczewska M, Zabierowska Slusarczyk G, Lee Y (2004) Method validation for HPLC analysis of related substances in pharmaceutical drug products. In: Chan CC, Lam H, Lee YC, Zhang XM (eds) Analytical method validation and instrument performance verication. Wiley, New York, pp 2748 7. Skoog D, Holler F, Nieman T (1998) High performance liquid chromatography,

principles of instrumental analysis, 5th edn. Saunders College Publishing, Philadelphia, pp 725726 8. United States Pharmacopeial Convention (2005) The United States pharmacopeia, USP NF 29, High performance liquid chromatography. The United States Pharmacopeial Convention, pp 26442651 9. United States Pharmacopeial Convention (2005) The United States Pharmacopeia, USP NF 29, Validation of compendial methods. The United States Pharmacopeial Convention, pp 30503053


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