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Chapter-5
Stability Indicating Analytical Method DevelopmentStability Indicating Analytical Method DevelopmentStability Indicating Analytical Method DevelopmentStability Indicating Analytical Method Development
and Validation for theand Validation for theand Validation for theand Validation for the DDDDetermination of Cholecalciferoletermination of Cholecalciferoletermination of Cholecalciferoletermination of Cholecalciferol
in Alendronate Sodium and Cholecalciferol Tabletsin Alendronate Sodium and Cholecalciferol Tabletsin Alendronate Sodium and Cholecalciferol Tabletsin Alendronate Sodium and Cholecalciferol Tablets
by HPC!by HPC!by HPC!by HPC!
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1. Introduction:
Alendronate sodium and Cholecalciferol Drug product [1-5] is a bisphosphonate and
vitamin D combination available with the Brand name Fosamax plus D. It works by
slowing bone breakdown and allowing new bone to be formed. It also helps to maintain
adequate levels of vitamin D in the body which is required for bone formation.
Cholecalciferol (vitamin D3) is a secosterol,which is the natural precursor of the calcium-
regulating hormone calcitriol (1,25dihydroxyvitamin D3). Cholecalciferol is a white,
crystalline, odorless powder. Cholecalciferol is practically insoluble in water, freely
soluble in usual organic solvents, and slightly soluble in vegetable oils.
Fig5.1:Chemical structure of Cholecalciferol
Chemical name :(5Z,7E)-9,10-secocholesta-5,7,10(19)-trien-3b-ol.
Moleculr formula: C27H44O
Moleculr Weight: 384.6
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Vitamin D3 (Cholecalciferol) is an essential nutrient, required for calcium absorption and
healthy bones. Vitamin D3 is produced in the skin by photochemical conversion of 7-
dehydrocholesterol to previtamin D3 by ultraviolet light. This isfollowed by non-
enzymatic isomerization to vitamin D3. Vitamin D3 in the skin and dietary vitamin D3
(absorbed into chylomicrons) is converted to 25-hydroxyvitamin D3 [6-9] in theliver.
Conversion to the active calcium-mobilizing hormone1,25-dihydroxyvitamin D3
(calcitriol) in the kidney is stimulated by bothparathyroid hormone and
hypophosphatemia. The principal action of1,25-dihydroxyvitamin D3 is to increase
intestinal absorption of bothcalcium and phosphate as well as regulate serum calcium,
renalcalcium and phosphate excretion, bone formation and bone resorption.Vitamin D is
required for normal bone formation. Vitamin Dinsufficiency develops when both sunlight
exposure and dietaryintake are inadequate. Insufficiency is associated with
negativecalcium balance, increased parathyroid hormone levels, bone loss,and increased
risk of skeletal fracture. In severe cases, deficiencyresults in more severe
hyperparathyroidism, hypophosphatemia,proximal muscle weakness, bone pain and
osteomalacia.
FOSAMAX PLUS D Tablets for oral administration contains 91.37 mg of alendronate
monosodium salt trihydrate, the molar equivalent of70 mg of free acid, and 70 mcg of
cholecalciferol equivalent to2800 International Units (IU) vitamin D and another strength
with 5600 International Units (IU) vitamin D.
Alendronate sodium and cholecalciferol tablets contain thefollowing inactive ingredients:
microcrystalline cellulose, lactoseanhydrous, medium chain triglycerides, gelatin,
croscarmellose sodium,sucrose, colloidal silicon dioxide, magnesium stearate,
butylatedhydroxytoluene, modified food starch, and sodium aluminum silicate.
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1. Literature survey and Scope of the study:
Cholecalciferol is light sensitive and undergoes oxidation if exposed to normal
environmental conditions. Hence Cholecalciferol API is packaged under argon gas, at 2-
8C and should be protected from light. It is soluble in ethanol, acetone, ether,and
chloroform. Practically insoluble in water. Solutions in propylene glycol or corn oil retain
the potency over long periods of time at 40C.
Cholecalfiferol solutions in volatile solvents are unstable and should be used
immediately.In solution state cholecalciferol converts to pre cholecalciferol with the time.
Phamacological activity is due to both cholecaciferol and pre cholecalciferol [1-5 &
10].Since cholecalciferol is light sensitive and easily umdergoes oxidation, it should be
properly protected in the drug products. To stabilize cholecalciferol it is mixed with
medium chain triglycerides, gelatin and Butylated hydroxyl toluene. Due to this type of
protection it is very difficult to extract cholecaliferol from the drug products.
There were few methods reported in the literature and all are related to the raw material
assay analysis [11-14], but none of them discussed about specificity of the method and the
extraction of Cholecalciferolfrom the stabilized formulations. This drug product is not
official in USP or any other pharmacopoeias. By considering all the complexities attempts
were made to develop [15-16] simple and stability indicating validated [17-19] Assay
method for the routine testing and stability analysis of cholecalciferol formulations.
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3.Development and Validation of a novel stability indicating HPLC Assay method for
determination of Cholecalciferolin Alendronate sodium and cholecalciferolTablets
3.1 Experimental
3.1.1 Materials and Reagents
Alendronate sodium and cholecalciferol tablets, Placebo, Working standard and
impurities were received from Dr. Reddys Laboratories, Hyderabad, India. Ethanol, n-
Pentanoland n-hexanesolvents were purchased from Merck. Sodium chloride and
Butylated hydroxyl toluene solid chemicals were purchased from Merck. Ultra-pure
water (resistivity 18.2Mcm) collected by a Milli-Q system (Millipore, Milford, MA,
USA).
3.1.2 Equipments
The Liquid chromatographic system used was Waters 2695 quaternary pump plus
autosampler and a 2996 photo diode array detector (Waters Corporation, 34Maple Street,
Milford, MA, 01757 USA). The output signal wasmonitored and processed using
empower software on Pentiumcomputer (Digital equipment Co) and empower
software.Cintex digital water bath was used for specificity study. Stability studies were
carried out in humidity chamber(Thermo lab humidity chamber, India) and photo stability
studies werecarried out in a photo stability chamber (SUNTEST XLS+, ATLAS,
Germany). Thermal stability studies were performedin a Thermolab hot air oven.
3.1.3 Preparation of Diluent
n-Hexane was used as a diluent.1% w/w Butylated hydroxyl toluene in ethanol, 0.5% w/v
NaCl solution were used in sample preparation.
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temperatureon the tailing factor of Cholecalciferol peak, resolution between pre
Cholecalciferol and trans-cholecalciferoland %RSD for peak areas of replicate injections
of standard was studied.Flow rates of 1.8 mL min-1
and 2.2 mL min-1
and column
temperatures of 20C and 30C were studied during this study.
3.2 Results and Discussion
3.2.1: Optimization of sample preparation:
Cholecalciferol is sensitive towards light and high temperatures.Generally it is stabilized
with the excipients, which prevents the direct exposure of cholecalciferol and imparts
stability to the drug product. Extraction of cholecalciferol from this gelation matrix was
critical step for the method development. Crushed tablet powder was dispersed in water
and heated at 50C for 30 minutes, this allows the gelatin to swell and releases drug
slowly. Cholecalciferol released from the gelatin matrix was suspended in water as it is
insoluble in water. To get the cholecalciferol in to the solution state ethanol and BHT
solution was added. After addition of ethanol cholecalciferol dissolves in the ethanol-
water mixture.BHT solution stabilizes the drug in the solution state.Cholecalciferol is
extracted to the hexane layer by liquid-liquid extraction method. NaCl solution was used
to saturate the aqueous layer such that cholecalciferol will be thrown to the hexane layer.
Critical steps optimized during sample preparation:
Cholecalciferol drug release from the Tablets matrix.
Solubilisation and stabilization of cholecalciferol.
Liquid-liquid extraction with 100% accuracy.
3.2.2Optimization of chromatographic conditions:
The main objective of the chromatographic separation is to get good separation for
Cholecalciferol and Precholecalciferol from the other impurities.
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Wave length maximum for cholecalciferol is about 265 nm, whereas for pre
cholecalciferol it is about 259 nm. As the cholecaldiferol is the main component, 265 nm
was selected for the quantification. To nullify the differences in UV absorbance,
conversion factor determined for cholecalciferol to precholecalciferol. As the
pharmacological activity is due to both the components,sum of cholecalciferol and
precholecalciferol peaks considered for Assay calculation.
Fig 5.2: UV spectra of precholecalciferol and cholecalciferol; 9.665 peak is pre
cholecalcifrol and 18.114 peak is cholecalciferol.
Cholecalciferol degradation impurity, Trans-cholecalciferol is elutes very close to the
precholecalciferol. To ensure the separation of this impurity,resolution solution injection
was given as part of system suitability. This ensures the specificity of the method for every
HPLC sample set.Transcholecalciferol impurity can be generated by heating the
cholecalciferol solution.
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Optimized chromatographic conditions:
Detector: Liquid Chromatographic system equipped with UV Visible detector at 265 nm
Column :Hypersil Silica, 250 mm x 4.6 mm, 5 m
Mobile phase :n-pentanol and n-hexane in the ratio of3:997 (v/v) respectively
Flow rate :2 mL min-1
Column temperature :50C
Injection volume :100L
Run time :25 minutes
Fig5.3: Typical chromatogram of Blank
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Fig5.6: Typical chromatogram of Test preparation
3.2.3Specificity:
Forced degradation studies were conducted to ensure the stability indicating nature of the
method. As the cholecalciferol is insoluble in water the dispersed portion of
cholecalciferol is extracted with the help of ethanol, and treated with the different types of
stress reagents.
After exposing to stress conditions, samples were prepared as per test preparation.
Stressed samples were injected into the HPLC system with PDA detector by following test
method conditions. All degradant peaks were resolved from cholecalciferol and pre
cholecalciferol in the chromatograms of all stressed samples.Peak purity was evaluated for
cholecalciferol and pre cholecalciferol.
There was no purity flag for Cholecalciferol and pre Cholecalciferol in any stressed
samples. This indicates that there is no interference of degradation impurities.
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Table 5.1: Peak Purity Results of Forced Degradation Studies
Stress Condition Peak purity results
Name Purity
angle
Purity
threshold
Purity flag
Refluxed with 1N HCl solution at100 C for 60 minutes.
Cholecalciferol 0.343 0.424 No
Precholecalciferol 1.212 1.352 No
Refluxed with 1N NaOH solution at
100 C for 2 hours.
Cholecalciferol 0.110 0.295 No
Precholecalciferol 0.518 0.736 No
Refluxed with 1% Hydrogen
peroxide solution at 100 C for 2
hours.
Cholecalciferol 0.317 0.595 No
Precholecalciferol 1.460 2.088 No
Refluxed with purified water at 100
C for 2 hours.
Cholecalciferol 0.047 0.214 No
Precholecalciferol 0.227 0.244 No
Exposed to UV light both at shorter
and longer wavelengths for about
200 watt-hours / square meter for
about 7 days.
Cholecalciferol 0.044 0.257 No
Precholecalciferol 0.804 0.244 No
Dry heated at 105 C for about
4hours.
Cholecalciferol 0.085 0.277 No
Precholecalciferol 0.804 0.952 No
Exposed to humidity at 25C, 90%
RH for about 7 days.
Cholecalciferol 0.028 0.241 No
Precholecalciferol 0.530 0.629 No
Exposed to Visible light for about
1.2 million lux
Cholecalciferol 0.101 0.318 No
Precholecalciferol 0.580 0.629 No
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Fig5.7: Typical Chromatogram of Acid stressed Test
Fig 5.8:Purity Plot of Acid stressed cholecalciferol
Fig 5.9: Purity Plot of Acid stressed Pre cholecalciferol
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Fig 5.10: Typical Chromatogram of Base stressed Test
Fig 5.11: Purity Plot of Base stressed Cholecalciferol
Fig 5.12: Purity Plot of Base stressed pre-cholecalciferol
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Fig 5.13: Typical Chromatogram of Peroxide stressed Test
Fig 5.14: Purity Plot of Peroxide stressed Cholecalciferol
Fig 5.15: Purity Plot of Peroxide stressed Precholecalciferol
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Fig 5.16: Typical Chromatogram of Aqueous stressed Test
Fig 5.17: Purity Plot of Aqueous stressed Cholecalciferol
Fig 5.18: Purity Plot of Aqueous stressed pre-Cholecalciferol
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Fig 5.19: Typical Chromatogram of UV light stressed Test
Fig 5.20: Purity Plot of UV light stressed Cholecalciferol
Fig 5.21: Purity Plot of UV light stressed Pre-Cholecalciferol
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Fig 5.22: Typical Chromatogram of Dry heat stressed Test
Fig 5.23: Purity Plot of Dry heat stressed Cholecalciferol
Fig 5.24: Purity Plot of Dry heat stressed pre Cholecalciferol
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Fig 5.25: Typical Chromatogram of Humidity stressed Test
Fig 5.26: Purity Plot of Humidity stressed Cholecalciferol
Fig 5.27: Purity Plot of Humidity stressed pre cholecalciferol
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Fig 5.28: Typical Chromatogram of visible light stressed Test
Fig5.29: Purity Plot of visible light stressed Cholecalciferol
Fig 5.30: Purity Plot of visible light stressedpre Cholecalciferol:
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3.2.4: Method validation results
3.2.4.1 Precision of test Method:
HPLC system precision was evaluated by injecting Cholecalciferolstandard solutionfive
times into the HPLC system. The liquid chromatographic system, suitability parameters
were evaluated.
Table 5.2: System Suitability
System suitability Observed value Acceptance
criteria70 mg/2800 IU 70 mg/5600 IU
Tailing factor for Cholecalciferolpeak from standard solution
1.0 1.0 NMT 2.0
Relative standard deviation for peak
area of Cholecalciferol from five
injections of standard
0.3% 0.4% NMT 2.0%
Resolution between Pre
Cholecalciferoland
Transcholecalciferol from system
suitability solution
2.0 2.0 NLT 1.0
Repeatability:
Repeatability of the assay method was studied by carrying out six independent assays of
test samples against qualified standard. Repeatability was performed on boththe strengths
of the drug product.% RSD of six consecutive assays was less than 1%. Resultshas shown
insignificant variation in measured response, which demonstrated that the method was
repeatable with lower % RSDs below the limit of 2.0.
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Table 5.3:Repeatability
S.No.
%Assay of Cholecalciferol
70 mg/2800 IU 70 mg/5600 IU
1 101.8 102.6
2 104.8 102.2
3 104.1 103.2
4 102.3 101.7
5 98.9 101.7
6 98.9 98.9
Average 101.8 101.1
RSD 2.3% 1.4%
Intermediate Precision:
Intermediate precision was performed by carrying out six independent assays of
Cholecalciferol against qualified standardover different days, different instruments, and
different columns. The mean % assay and % R.S.D for assay values were found to be 99.6
and 0.7 % respectively.
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Table 5.4: Repeatability and Reproducibility
Sample
No.
% Assay of Cholecalciferol
Repeatability Reproducibility
70 mg/2800 IU 70 mg/5600 IU 70 mg/2800 IU 70 mg/5600 IU
1101.8 102.6 102.4 101.9
2104.8 102.2 103.7 100.7
3104.1 103.2 101.3 100.9
4102.3 101.7 103.2 101.0
598.9 101.7 101.0 102.8
698.9 98.9 103.3 101.7
Mean101.8 101.1 102.0 101.6
RSD2.3% 1.4% 1.5% 0.8%
3.2.4.2Linearity:
Linearity was established by plotting a graph between concentrations versus area and
determined the correlation coefficient. A series of solutions of Cholecalciferol standard,
were prepared in the concentration range of 1.3944 ppm to 25.6571 ppm and analyzed as
per test method. A graph was plotted to concentration in ppm on X- axis versus peak
response on Y-axis. The detector response was found to be linear with a correlation
coefficient of 0.999.
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Table 5.5: Linearity
S.No.Concentration
(g/mL)
Peak Area
01 1.3944197675
02 1.9522 279956
03 2.7888398659
04 5.5776800808
05 11.15521623327
06 14.50182055090
07 16.73292410840
08 20.07942908456
09 22.31053126782
10 25.65713714820
Co-efficient of Correlation (r) : 0.999
Slope (m) : 143365
Intercept (b) : 435.53
% Bias at 100% response : 0
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Fig5.31: Linearity Graph of Cholecalciferol
3.2.4.3 Accuracy
Accuracy samples were prepared in triplicate at various concentrations ranging from 25%
to 450% (25%, 100%, 200%, 300%, 400% and 450%) of the target concentration. All
recovery samples were prepared in triplicate and injected in to the HPLC system.
y = 143365x + 435.53
R= 0.999
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
0 5 10 15 20 25 30
Linearity-Cholecalciferol
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Table 5.6: Accuracy
Sample
No.
Spike level mg added mg found % Recovery Mean %
Recovery
1
25%
0.035 0.035 98.8
98.62 0.035 0.034 98.3
3 0.035 0.035 98.6
1
100%
0.140 0.136 97.2
97.92 0.140 0.137 97.7
3 0.140 0.138 98.7
1
200%
0.280 0.280 99.9
98.82 0.281 0.275 97.8
3 0.281 0.277 98.61
300%
0.419 0.413 98.5
98.72 0.419 0.411 98.0
3 0.419 0.418 99.7
1
400%
0.560 0.559 99.8
99.72 0.561 0.558 99.5
3 0.560 0.559 99.9
1
450%
0.630 0.624 99.0
99.12 0.630 0.623 98.9
3 0.630 0.626 99.4
3.2.4.4Solution stability and Mobile phase stability:
A study to establish the stability of Cholecalciferol standard preparation and test
preparation in refrigerator was conducted at Initial, after 1 day, and 2 days. The assay of
Cholecalciferol test preparation and standard preparation were estimated against freshly
prepared standard each time. The difference in % assay of Test preparations from initial to
1 day, and 2 days was found to be within 2%, and similarity factor for standard from
initial to 1 day, and 2 days was 0.99.
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Table 5.7: Stability of Cholecalciferol Standard and Test Preparation
Time in
days
Standard
Similarity
factor
% Assay of test
preparationDifference
Test-1 Test-2 Test-1 Test-2
Initial NA 102.6 102.2 NA NA
1 0.99 102.3 102.7 0.3 0.5
2 0.99 102.1 99.4 0.5 2.8
A study to establish Bench top stability of mobile phase at initial, after 1 day, 3 days and 5
days was conducted.The system suitability parameters were evaluated as per the test
method and found to be within the limits. The difference in % assay from initial to 1 day,
2 days and 5 days was found to be within the limit of 3%.
Table5.8: Stability of Mobile Phase-System suitability results
SystemSuitability Parameters
Observed value Acceptance
CriteriaInitial Day-1 Day-3 Day-5
Tailing factor for Cholecalciferol
peak from standard solution
1.0 1.0 1.0 1.0 NMT 2.0
Relative standard deviation of
Cholecalciferol from five
injections of standard
0.4% 0.3% 0.2% 0.2% NMT 2.0%
Resolution between Pre
cholecalciferol and
Transcholecalciferol from system
suitability solution
2.0 2.0 2.1 2.0 NLT 1.0
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Table 5.9: Stability of Mobile Phase-Test preparation results
Time in days % Assay Difference from
initial
Test-1 Test-2 Test-1 Test-2
Initial 101.1 101.1 NA NA
1 101.1 101.4 0.0 0.3
2 98.8 98.8 2.3 2.3
5 102.2 102.8 1.1 1.7
3.2.4.5 Robustness:
In all the method variationslike flow rate and column temperature the tailing factor,
the % R.S.D for the Cholecalciferol peak area and resolution between precholecaliferol
and cholecaliferol from the five replicate injections of standard was found to be with in the
acceptable limits.This study indicates the robustness of the method for all the possible
chromatographic variations.
Table 5.10: Robustness-Flow variation:
System Suitability
Parameters
Observed value with Flow rate Acceptance
Criteria
1.8 mL/min 2.0mL/min 2.2mL/min
Tailing factor for
Cholecalciferol peak from
standard solution
1.2 1.2 1.1 NMT 2.0
Relativestandard deviation
of Cholecalciferol from
five injections of standard
0.1% 0.1% 0.2% NMT 2.0%
Resolution between Pre
Cholecalciferol and
Transcholecalciferol from
system suitability solution
2.0 1.9 1.9 NLT 1.0
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Table 5.11: Robustness-Temperature variation:
System Suitability Parameters
Observed value at column
temperature Acceptance
Criteria
20C 25C 30C
Tailing factor for Cholecalciferol
from standard solution 1.1 1.2 1.1 NMT 2.0
Relative standard deviation of
Cholecalciferol from five injections
of standard
0.2% 0.1% 0.4% NMT 2.0%
Resolution between Pre
Cholecalciferol
and Transcholecalciferol from
system suitability solution
2.2 1.9 1.8 1.0
3.3 Conclusion:
A simple, precise, cost effective and stability indicating Normal phase-HPLC Assay
method has been developed and validated for the determination of Cholecalciferol in
pharmaceutical formulations.This method is stability indicating by separating all the
possible degradation products from the Cholecalciferol peak within 25 minutes run
time.The method wascompletely validated as per ICH recommendations, showing
satisfactory data for all the methodvalidation parameters. The proposed method can be
used for the routine samples and stability samples analysis.This method can be used for
any cholecalciferol formulations with minimum method verification.
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ca8b-58cb0f65b5d9(tab)
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ICH: Q2A, Text on validation of analytical procedure (October 1994).
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