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172

Chapter-5

Stability Indicating Analytical Method DevelopmentStability Indicating Analytical Method DevelopmentStability Indicating Analytical Method DevelopmentStability Indicating Analytical Method Development

and Validation for the and Validation for the and Validation for the and 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 HPLC.by HPLC.by HPLC.by HPLC.

173

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

174

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.

175

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-

8°C 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 40°C.

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.

176

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. Reddy’s 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 18.2MΩ cm) 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.

177

3.1.4 Preparation of Standard Solution

The stock solution of Cholecalciferol(0.28 mg mL-1

) was prepared in Ethanol. The

standard solution (0.08 mg mL-1

) was obtained by liquid-liquid extraction to hexane layer.

3.1.5 Preparation of Sample solution

Ten Alendronate sodium and cholecalciferoltablets were crushed to fine powder with the

help of mortar and pestle. 0.14 mg equivalent of cholecalciferol tablets powder was taken

into 100 ml volumetric flask. Added about 15 ml of milli-Q water and kept on the shaking

water bath at 50° C for 30 minutes.After wards sonicated for about 10 minutes and then

cooled to room temperature.1 mL of BHT solution and 15 mL of ethanol was added and

then sonicated for 15 minutes.25 mL of n-hexaneand 1 mL of 0.5% NaClsolution was

added to the above flask and mixed well with the help of vertex mixer.Allow to stand at

room temperature for 5 minutes for the separation of two layers.

3.1.6 Specificity

Forced degradation studies were performed to demonstrate selectivity and stability

indicating capability of the method. The samples were analyzed and evaluated the peak

purity for Cholecalciferol and precholecalciferolby using photo diode array detector.

3.1.7 Method Validation

3.1.7.1 Precision

Repeatability of the assay method was studied by performing systemprecision, followed

by carrying out six independent assays of Cholecalciferol test samples against qualified

standard.The percentage R.S.D of six assay values obtained was calculated.

The intermediate precision of the method was also evaluated with different

HPLC systems and different HPLC columns in different days.

178

3.1.7.2 Linearity

Linearity test solutions for assay method wereprepared from stock solution, at ten

concentration levels in the range of about 1.4 to 25 µg mL-1

.Thepeak area versus

concentration were used to plot a graph and correlation co-efficient was determined.

3.1.7.3 Accuracy

A study of recovery of Cholecalciferol from drug product was conducted. Sample

solutions were prepared in triplicate by taking Alendronate sodium and Cholecalciferol

blend equivalent to about, 25%, 100%, 200%, 300%, 400% and 450% of the target test

concentration. The % recovery was calculated.

3.1.7.4Solution stability and Mobile phase stability

Test preparation contains highly volatile solvents n-hexane, hence solution stability

was performed in refrigerator. The sample solutions were storedin refrigerator at 2-8 °C in

tightly stoppered volumetric flasks, and analyzed after 1 day, and 2 days against freshly

prepared standard solution each time. For sample solutions, % assay and the percent

difference in assay between initial and each interval was calculated. Similarity factor was

calculated for stored standard solution to the freshly prepared standard solution.

Mobile phase stability was carried out by assaying freshly prepared sample solutions

against freshly prepared standard solutions after 1 day, 2 days and 5 days interval. Mobile

phase is kept constant during the study period. The % assay of Cholecalciferol was

calculated for the study period during mobile phase stability and solution stability

experiments.

3.1.7.5 Robustness

To determine the robustness of the method experimental conditions were purposely altered

and evaluated the system suitability parameters. Five replicate injections of standard

solutions are injected under each parameter change. The effect of flow rate and column

179

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 20ºC and 30ºC 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 50ºC 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.

180

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.

181

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 µm

Mobile phase :n-pentanol and n-hexane in the ratio of3:997 (v/v) respectively

Flow rate :2 mL min-1

Column temperature :50°C

Injection volume :100µL

Run time :25 minutes

Fig5.3: Typical chromatogram of Blank

182

Fig5.4: Typical chromatogram of Standard

Fig5.5: Typical chromatogram of system suitability solution

183

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.

184

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 at

100° 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.


0.295

No


Refluxed with 1% Hydrogen

peroxide solution at 100° C for 2

hours.


0.595

No


Refluxed with purified water at 100°

C for 2 hours.


0.214

No


Exposed to UV light both at shorter

and longer wavelengths for about

200 watt-hours / square meter for

about 7 days.


0.257

No


Dry heated at 105° C for about

4hours.


0.277

No

Precholecalciferol 0.804

0.952 No

Exposed to humidity at 25°C, 90%

RH for about 7 days.


0.241

No


Exposed to Visible light for about

1.2 million lux


0.318

No


185

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

186

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

187

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

188

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

189

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

190

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

191

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

192

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:

193

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

criteria

70 mg/2800 IU 70 mg/5600 IU

Tailing factor for Cholecalciferol

peak 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.

194

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.

195

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

1 101.8 102.6 102.4

101.9

2 104.8 102.2 103.7

100.7

3 104.1 103.2 101.3

100.9

4 102.3 101.7 103.2

101.0

5 98.9 101.7 101.0

102.8

6 98.9 98.9 103.3

101.7

Mean 101.8 101.1 102.0

101.6

RSD 2.3% 1.4% 1.5%

0.8%

3.2.4.2 Linearity:

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.

196

Table 5.5: Linearity

S.No. Concentration

(µg/mL)

Peak Area

01 1.3944 197675

02 1.9522 279956

03 2.7888 398659

04 5.5776 800808

05 11.1552 1623327

06 14.5018 2055090

07 16.7329 2410840

08 20.0794 2908456

09 22.3105 3126782

10 25.6571 3714820

Co-efficient of Correlation (r) : 0.999

Slope (m) : 143365

Intercept (b) : 435.53

% Bias at 100% response : 0

197

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

198

Table 5.6: Accuracy

Sample

No.

Spike level “mg” added “mg” found % Recovery Mean %

Recovery

1

25%

0.035 0.035 98.8

98.6 2 0.035 0.034 98.3

3 0.035 0.035 98.6

1

100%

0.140 0.136 97.2

97.9 2 0.140 0.137 97.7

3 0.140 0.138 98.7

1

200%

0.280 0.280 99.9

98.8 2 0.281 0.275 97.8

3 0.281 0.277 98.6

1

300%

0.419 0.413 98.5

98.7 2 0.419 0.411 98.0

3 0.419 0.418 99.7

1

400%

0.560 0.559 99.8

99.7 2 0.561 0.558 99.5

3 0.560 0.559 99.9

1

450%

0.630 0.624 99.0

99.1 2 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.

199

Table 5.7: Stability of Cholecalciferol Standard and Test Preparation

Time in

days

Standard

Similarity

factor

% Assay of test

preparation Difference

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

System

Suitability Parameters

Observed value Acceptance

Criteria

Initial

Day-1 Day-3 Day-5


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%


cholecalciferol and

Transcholecalciferol from system

suitability solution

2.0

2.0

2.1

2.0

NLT 1.0

200

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%


Cholecalciferol and

Transcholecalciferol from

system suitability solution

2.0

1.9

1.9

NLT 1.0

201

Table 5.11: Robustness-Temperature variation:

System Suitability Parameters

Observed value at column

temperature

Acceptance

Criteria

20°C

25°C

30°C


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%


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.

202

References

1. http://www.drugs.com/ppa/alendronate-sodium-cholecalciferol.html

2. http://dailymed.nlm.nih.gov/dailymed/lookup.cfm?setid=c0ec7e75-03b9-4b5b-

ca8b-58cb0f65b5d9(tab)

3. http://www.rxlist.com/fosamax-plus-d-drug.htm

4. http://www.fda.gov/Safety/MedWatch/SafetyInformation/ucm208690.htm

5. http://www2.shimadzu.com/applications/LC/Shimadzu_Analysis_Vitamins_2007.

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Scand J Clin Lab Invest Suppl. 1997;227:35–45.

9. Eisman JA, Shepard RM, DeLuca HF. Anal Biochem. 1977;80:298–305.

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11. Grant A. Abernethy ;AnalBioanalChem (2012) 403:1433-1440.

12. Pelc B, Marshall DH. Steroids. 1978 Jan;31(1):23-9

13. Takada K. J Steroid Biochem. 1983 Apr;18(4):507-9.

14. Katherine M. Phillips,W. Craig Byrdwell, Jacob Exler, James M. Harnly, Joanne

M. Holden ichael F. Holick , Bruce W. Hollis. Journal of Food Composition and

Analysis 21 (2008) 527

15. A.Braithwaite, F.J.Smith. Chromatographic Methods, Fifth edition, Kulwer

Academic publishers, Dordrecht-Netherlands, 1999, 258-354.

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16. LioydR.Snyder, Joseph J.Kirckland, Loseph.L.Glaich. Practical HPLC Method

Development, second edition, John Wiley & Sons Inc, United states of America,

1997,1-265.

17. ICH: Q2A, Text on validation of analytical procedure (October 1994).

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19. ICH Q2 (R1), Validation of Analytical Procedures Text and Methodology

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