172

Chapter-5









Stability Indicating Analytical Method Development Stability Indicating Analytical Method Development Stability Indicating Analytical Method Development Stability Indicating Analytical Method Development
and Validation for the and Validation for the and Validation for the and Validation for the D DD Determination of Cholecalciferol etermination of Cholecalciferol etermination of Cholecalciferol etermination of Cholecalciferol
in Alendronate Sodium and Cholecalciferol Tablets in Alendronate Sodium and Cholecalciferol Tablets in Alendronate Sodium and Cholecalciferol Tablets in Alendronate Sodium and Cholecalciferol Tablets
by HPC! by HPC! by HPC! by HPC!




















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: C
27
H
44
O
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-
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.





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. 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 18.2M 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 g 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 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.
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 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


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.
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











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
(g/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

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






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%
Resolution between Pre
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
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.



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.
pdf
6. Andrew M WoottonClinBiochem Rev. 2005 February; 26(1): 3336
7. Sackrison JL, Ersfield DL, Miller AB, Olson GT, MacFarlane GD. Clin Chem.
2002;48:A122
8. Schmidt-Gayk H, Bouillon R, Roth HJ, Seamark DA, Trafford DJ, Makin HL.
Scand J Clin Lab Invest Suppl. 1997;227:3545.
9. Eisman JA, Shepard RM, DeLuca HF. Anal Biochem. 1977;80:298305.
10. Karen C. Scott, J.DavidLatshaw, Animal Feed Science and Technology 47 (1) ;
99-105
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.
203

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).
18. ICH: Q2B, Analytical Validation Methodology (November 1996).
19. ICH Q2 (R1), Validation of Analytical Procedures Text and Methodology
(November 2005).