UDCA 250 mg capsules Document code: 3.2.P.5.

2 2015/ 00
UDCA 500 mg capsules Date: September 2015

3.2.P.5.2. Analytical procedures

CAPSULE

1. Capsule appearance
Determined by means of an organoleptic examination.

2. Average mass
Determined according to Ph.Eur. current edition method 2.9.5.

3. Uniformity of dosage unit (mass variation)

Determined according to Ph.Eur. current edition method 2.9.40.

4. Dissolution test
• Equipment: dissolver with paddle stirrer and suitable sinker
• Medium: phosphate buffer solution pH 8.4
• Temperature: 37°C
• Volume: 900 ml
• Speed: 50 rpm
• Content determination method: HPLC

Preparation of buffer solution pH 8.4 (dissolution medium)

In a 1000 ml calibrated flask mix 250 ml of KH2PO4 solution 0.2M and 280 ml of KOH solution
0.2M. Adjust to pH 8.4 with KOH solution 0.2M and dilute to volume with purified water.

Preparation of sample solution

Immerse one capsule in the dissolution medium in each container and wait for one minute
between each capsule and the next one.
Once the necessary time (45 minutes) has elapsed since the beginning of the test, take
approximately 10 ml of each sample following the capsule adding sequence.
The sampling must come from an intermediate area between the surface of the dissolution
medium and the upper part of the stirring blade.
The sampling solution must be immediately filtrated with a 0.45 µm membrane filter.

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UDCA 250 mg capsules Document code: 3.2.P.5.2 2015/ 00
UDCA 500 mg capsules Date: September 2015

For UDCA 500 mg capsules only:

Into a 10 ml calibrated flask transfer 5 ml of the previous filtrated sampling solution and dilute to
volume with dissolution medium. Filtrate with a 0.45 µm membrane filter.

Preparation of Primary standard solution

Weigh in a 10 ml calibrated flask approximately 27.8 mg of standard Ursodeoxycholic acid.
Dissolve in 5 ml of ethanol and dilute to volume with dissolution medium.
Into a 10 ml calibrated flask transfer 1.0 ml of this solution and dilute to volume with dissolution
medium. Filtrate with a 0.45 µm membrane filter.
(Theoretic concentration = 0.278 mg/ml)

Preparation of Secondary standard solution

Weigh in a 10 ml calibrated flask approximately 27.8 mg of standard Ursodeoxycholic acid.
Dissolve in 5 ml of ethanol and dilute to volume with dissolution medium. Into a 10 ml calibrated
flask transfer 1.0 ml of this solution and dilute to volume with dissolution medium. Filtrate with a
0.45 µm membrane filter.
(Theoretic concentration = 0.278 mg/ml)

Equipment
HPLC Chromatograph with RI detector and auto-sampler

Chromatographic conditions
(Same chromatographic conditions used in Ursodeoxycholic acid content par. 5.2.9)
- Column: C18 - 250 x 4 mm - 5 µm (Lichrosphere C18 or equivalent)
- Mobile phase: KH2PO4 0.075 M pH 3.0/ Acetonitrile (50:50 v/v)
- Column temperature: 40°C
- Flow: 1.0 ml/min
- Detector: Refractive index
- Detector temperature: 37°C
- Run time: 15 minutes
- Injection volume: 50 µl

Preparation of KH2PO4 0.075 M pH 3.0

In a 1000 ml calibrated flask weight 10.2068 g of KH2PO4, add 900 ml of purified water and
dissolve. Adjust the pH of the solution to 3.0 using Phosphoric acid 85%. Dilute to volume with
purified water.

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UDCA 250 mg capsules Document code: 3.2.P.5.2 2015/ 00
UDCA 500 mg capsules Date: September 2015

Procedure
Inject a suitable volume (50µl) of Primary standard solution and record the peak response. The
relative standard deviation for six replicate injections (areas) is not more than 2.0%.
Inject equal volume of Secondary standard solution and record the peak response. If relative
percentage standard deviation between the two standard solution is more than 1.0% prepare a
third standard solution.
Then inject equal volumes of each sample solution, record the chromatograms and measure the
areas of the main peak.
The tailing factor of Ursodeoxycholic acid in both standard solutions must be comprised between
0.8 and 1.5.

Calculation
Calculate the quantity of Ursodeoxycholic acid in the sample, expressed in %, by the formula:
sA x stW x stT% x cpsAW
Ursodeoxycholic acid % = ---------------------------------- x 9
stA x cpsW x 250
Where:
stT% = Standard Ursodeoxycholic acid Assay expressed as percentage
sA = Ursodeoxycholic acid area in the sample solution
stA = Ursodeoxycholic acid average area in the primary standard solution
stW = Standard Ursodeoxycholic acid weighing in mg
cpsW = Capsule sample weighing in mg
cpsAW = average weight in mg calculated on 20 capsules

Acceptance limit
The release of the active substance must be at least 65% within 45 minutes.

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UDCA 250 mg capsules Document code: 3.2.P.5.2 2015/ 00
UDCA 500 mg capsules Date: September 2015

5. Microbial purity tests

Proceed according to the following European Pharmacopoeia methods:
• Method 2.6.12“Microbiological examination of non-sterile products: total viable aerobic count”
• Method 2.6.13 “Microbiological examination of non-sterile products: tests for specified micro-
organisms”

TAMC and TYMC

Dissolve 10 g of sample in 100 ml of buffered peptone (final volume) and mix. Subsequently
introduce the suspension in waterbath at about 45°C for 5 minutes; add 10 ml of 1:10 dilution to
100 ml (final volume) of buffered peptone water. Perform the count method by seeding for each
medium 10 ml of this last solution (2ml on 5plates) and incubate TSA medium at 30-35°C for 3-5
days and SDA medium at 20-25°C for 5-7 days.
TAMC - TYMC (cfu/g): Sum the number of colony former units in respective plates and multiply
the result for 10. In case of absence of colonies express the result as <10cfu/g.

Detection of E. coli
Transfer 10 ml of the dilution 1:10 of the sample, previously prepare, in 100 ml of TSB and
incubate at 30°-35°C for 18-24 h.
After incubation, transfer 1 ml to 100 ml of Mac Conkey Broth and incubate at 42-44°C for 24-
48h, subculture on a plate of Mac Conkey Agar and incubate at 30-35°C for 18-72h.

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UDCA 250 mg capsules Document code: 3.2.P.5.2 2015/ 00
UDCA 500 mg capsules Date: September 2015

POWDER

6. Powder appearance
Determined by means of an organoleptic examination.

7. Water content (KF)

Determined according to Ph.Eur. current edition method 2.5.12.

Acceptance limits
The water content is not more than 3.0%.

8. Ursodeoxycholic acid Identification tests:

8.1 TLC Identification

Chromatographic conditions
Stationary phase Silica gel plate 60F254, layer thickness: 0.25 mm
Mobile phase Chloroform / Acetone / Acetic acid glacial (70:20:5)
Chromatographic run 15 cm
Detection Phosphomolybdic reactive 5%
Saturation time 2 hour

Phosphomolybdic reactive 5%
Dissolve 5 g of Phosphomolybdic acid in 95 ml of Acetic acid glacial and 5 ml of Sulfuric acid
concentrated.

Reference solution
Weight exactly 100.0 mg of Ursodeoxycholic acid into a 10 ml calibrated flask; dissolve and dilute
with methanol.
(Theoretic concentration = 10 mg/ml)

Test sample solution

Weigh 650.0 mg of powder into a 50 ml calibrated flask and dilute to volume with methanol.
Stir with magnetic stirrer for 5 minutes then allow to decant.
The sampling must be carried out on the clear supernatant.

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UDCA 500 mg capsules Date: September 2015

Procedure
The following quantities are applied to the plate:
Reference solution = 50 µl equivalent to 500 µg
Test sample solution = 50 µl equivalent to 500 µg
After applying the solutions 2 cm from the lower plate border, subject the plate to
chromatographic separation in the indicated eluent. Then allow the plate to dry in the air and
spray it homogeneously with the Phosphomolybdic reagent.
Place the plate in a stove at 120°C for 10 minutes to bring out the spots.

Results
The identification is positive when the sample solution shows a main spot corresponding to
Ursodeoxycholic acid, similar for size and Retention factor (Rf) to the spot in the Ursodeoxycholic
acid reference solution.
Rf of Ursodeoxycholic acid is 0.51.

8.2 HPLC Identification

Perform this identification test during the Ursodeoxycholic acid HPLC assay test (par.9).
Compare the sample chromatogram with that of the standard solution.
The identification is positive when the sample chromatogram exhibits a peak with retention time
and shape similar to that of the main peak of the standard solution chromatogram.

9. Ursodeoxycholic acid content (HPLC)

Equipment
HPLC Chromatograph with RI detector and auto-sampler

Chromatographic conditions
(Same chromatographic conditions used in Ursodeoxycholic acid dissolution test par. 5.2.4)
- Column: C18 250 x 4 mm 5 µm (Lichrosphere C18 or equivalent)
- Mobile phase: KH2PO4 0.075 M pH 3.0/ Acetonitrile (50:50 v/v)
- Column temperature: 40°C
- Flow: 1.0 ml/min
- Detector: Refractive index
- Detector temperature: 37°C
- Run time: 15 minutes
- Injection volume: 50 µl

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UDCA 500 mg capsules Date: September 2015

Preparation of Mobile phase

- Preparation of buffer solution (KH2PO4 0.075 M pH 3.0)
In a 1000 ml calibrated flask weight 10.2068 g of KH2PO4, add 900 ml of purified water and
dissolve. Adjust the pH of the solution to 3.0 using Phosphoric acid 85%. Dilute to volume with
purified water.
- Mix buffer solution with Acetonitrile (50:50 v/v)

Preparation of sample solution

Operate in triple.
Into a 100 ml calibrated flask weigh approximately 361.4 mg of the powder (equivalent to 278
mg of Ursodeoxycholic acid), dissolve with 50 ml of ethanol 96%, then dilute to volume with
buffer solution pH 8.4. Transfer 1.0 ml of this solution into a 10 ml calibrated flask and dilute to
volume with dissolution medium. Filtrate with a 0.45 µm membrane filter.
(Theoretic concentration is 0.278 mg/ml).

Preparation of buffer solution pH 8.4 (dissolution medium)

In a 1000 ml calibrated flask mix 250 ml of KH2PO4 solution 0.2M and 280 ml of KOH solution
0.2M. Adjust to pH 8.4 with KOH solution 0.2M and dilute to volume with purified water.

Preparation of Primary standard solution

(same solution used in Ursodeoxycholic acid dissolution test par. 5.2.4)
Weigh accurately in a 10 ml calibrated flask approximately 27.8 mg of standard Ursodeoxycholic
acid. Dissolve in 5 ml of ethanol and dilute to volume with dissolution medium. Into a 10 ml
calibrated flask transfer 1.0 ml of this solution and dilute to volume with dissolution medium.
Filtrate with a 0.45 µm membrane filter.
(Theoretic concentration = 0.278 mg/ml)

Preparation of Secondary standard solution

(same solution used in Ursodeoxycholic acid dissolution test par. 5.2.4)
Weigh accurately in a 10 ml calibrated flask approximately 27.8 mg of standard Ursodeoxycholic
acid. Dissolve in 5 ml of ethanol and dilute to volume with dissolution medium. Into a 10 ml
calibrated flask transfer 1.0 ml of this solution and dilute to volume with dissolution medium.
Filtrate with a 0.45 µm membrane filter.
(Theoretic concentration = 0.278 mg/ml)

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UDCA 250 mg capsules Document code: 3.2.P.5.2 2015/ 00
UDCA 500 mg capsules Date: September 2015

Procedure
Inject a suitable volume (50µl) of Primary standard solution and record the peak response. The
relative standard deviation for six replicate injections (areas) is not more than 2.0%.
Inject equal volume of Secondary standard solution and record the peak response. If relative
percentage standard deviation between the two standard solutions is more than 1.0% prepare a
third standard solution.
Then inject equal volumes of each sample solution, record the chromatograms and measure the
areas of the main peak. The relative standard deviation between the three injections is not more
than or equal to 2.0%
The tailing factor of Ursodeoxycholic acid in both standard solutions must be comprised between
0.8 and 1.5.

Calculation
Calculate the quantity of Ursodeoxycholic acid in the sample UDCA 250 mg capsules, expressed in
%, by the formula:
sA x stW x stT% x cpsAW
Ursodeoxycholic acid % = ----------------------------------
stA x cpsW x 25

Calculate the quantity of Ursodeoxycholic acid in the sample UDCA 500 mg capsules, expressed in
%, by the formula:
sA x stW x stT% x cpsAW
Ursodeoxycholic acid % = ----------------------------------
stA x cpsW x 50
Where:
stT% = Standard Ursodeoxycholic acid assay expressed as percentage
sA = Ursodeoxycholic acid area in the sample solution
stA = Ursodeoxycholic acid average area in the primary standard solution
stW = Standard Ursodeoxycholic acid weighing in mg
cpsW = Capsule sample weighing in mg
cpsAW = average net weight in mg calculated on 20 capsules

Acceptance limit
The resulting Ursodeoxycholic acid content must be included between 95.0 – 105.0 % (equals to
237.5 – 262.5 mg for UDCA 250 mg cps and 475 – 525 mg for UDCA 500 mg cps) of labelled
amount.

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UDCA 250 mg capsules Document code: 3.2.P.5.2 2015/ 00
UDCA 500 mg capsules Date: September 2015

10. Ursodeoxycholic acid content - Potentiometric titration (not routinary test):

Preparation of sample solution

Operate in triple.
Weight accurately approximately 397.8 mg of powder (equivalent to 306 mg of ursodeoxycholic
acid) into a 100 ml beaker. Add 60 ml of ethanol 80% v/v and sonicate for about 2 minutes.

Preparation of blank solution

Transfer 60 ml of Ethanol 80% v/v into a 100 ml beaker.

Procedure
Titrate with 0.1N NaOH, determining the end-point potentiometrically.
1 ml of 0.1N NaOH is equivalent to 39.26 mg of Ursodeoxycholic acid.
Titrate the blank solution and then the three sample solutions.

Calculation
Calculate the quantity of Ursodeoxycholic acid in the sample, expressed in titre %, by the
formula:

(sV-bV) x F x 39.26 x cpsAW

Ursodeoxycholic acid % = --------------------------------------- x 100
cpsW x D

sV= ml of NaOH used to sample titration

bV= ml of NaOH used to blank titration
F= titre of NaOH
cpsW = Powder sample weighing in mg
cpsAW = average net weight in mg calculated on 20 capsules
D= theoretical Ursodeoxycholic acid content/caps equivalent to 250 for UDCA 250 mg and 500 for
UDCA 500 mg

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UDCA 500 mg capsules Date: September 2015

11. Related substances (HPLC):

The known impurities of Ursodeoxycholic acid are:
- Impurity A (Ph. Eur.) : Chenodeoxycholic acid
- Impurity C (Ph. Eur) : Lithocholic acid

Equipment
HPLC Chromatograph with RI detector and auto-sampler

Chromatographic conditions
- Column: C18 - 100 x 4.6 mm - 3 µm (Luna C18 or equivalent)
- Mobile phase: Acetic acid solution 0.1%/ Methanol (20:80 v/v)
- Column temperature: 40°C
- Flow: 0.9 ml/min
- Detector Temperature: 37°C
- Run time: 20 minutes
- Injection volume: 50 µl

Preparation of sample solution

Into a 50 ml calibrated volumetric flask weigh accurately approximately 585 mg of the powder
(equivalent to 450 mg of Ursodeoxycholic acid), dissolve and dilute to volume with methanol. (A
solution)
Into a 10 ml calibrated volumetric flask transfer 5 ml of A solution and dilute to volume with
mobile phase. Filtrate with a 0.45 µm membrane filter.
(Theoretic concentration = 4.5 mg/ml)

Preparation of Reference solution A

In a 100 ml calibrated flask transfer 1.0 ml of A solution and dilute to volume with mobile phase.
Filtrate with a 0.45 µm membrane filter.
(Theoretic concentration is 0.045 mg/ml equivalent to 1.0 % of sample solution)

System suitability solution

Weight exactly 18 mg of Cholic acid into a 200 ml calibrated Volumetric flask, dissolve and dilute
to volume with methanol. Transfer 1 ml of this solution and 5 ml of A solution into a 10 ml
calibrated flask, dilute to volume with mobile phase. Filtrate with a 0.45 µm membrane filter.

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UDCA 500 mg capsules Date: September 2015

System Suitability
Resolution between the peaks due to Cholic acid and Ursodeoxycholic acid must be higher than
1.5.

Procedure
Inject a suitable volume (50 µl) of Sample Solution and record the peak responses. Then inject
equal volumes of Blank solution, System Suitability solution and Reference solution A.
In the sample solution not consider peaks due to blank solution.
Operating in these conditions the relative retention times (RRT) of known impurities respect to
Ursodeoxycholic acid peak (about 3 min) are:
RRT Chenodeoxycholic acid = 2
RRT Lithocholic acid =3.3

• Calculation
- Calculate the quantity of Known and unknown Impurities in the sample, expressed in %
Impurity by the formula:
IA x Fc
T%sd = ---------------
stA
Where:

IA = Each known and unknown impurity area in the sample solution

stA = Ursodeoxycholic acid area in the Reference solution A
Fc (correction factor Chenodeoxycholic acid and Lithocholic acid) = 1.1

• Acceptance limit

Impurities Limits
Impurity A ≤ 1.0%
Impurity C ≤ 0.1%
Any unspecified Impurity ≤ 0.1%
Total impurities ≤ 1.5%

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