LAB 7: STABILITY OF INJECTIONS
Shenyang Pharmaceutical University
PHARMACEUTICS III
LABORATORY 7: Investigation on the stability of 5%
Vitamin C injectable solution
1. LABORATORY OBJECTIVES
a) To understand the main factors influencing the stability of Vitamin C (Vc) injection.
b) To know the general methods used to test the stability of formulations during the
design phase.
2. INTRODUCTION
The basic quality requirements of pharmaceutical preparations are safety,
effectiveness, and stability. The decomposition of a drug substance not only reduces the
efficacy but also can cause toxic side effects by the degradation products. Therefore, the
stability of a pharmaceutical product is critical to ensure its safety and effectiveness. The
stability of an injection is of special importance because of the fact that injecting a
decomposed solution into the human body can be very dangerous. Depending on the
chemical structures of the drug, degradation mechanism can be different for different drugs.
Among the potential degradation mechanisms, hydrolysis and oxidation are two main
degradation pathways.
Vitamin C can be easily oxidized due to the presence of two enol group next to the
carbonyl group in the molecular structure. This oxidation process is extremely complicated.
In the aerobic conditions, it is firstly dehydrogenated to form dehydroascorbic acid which
is then hydrolyzed to form 2, 3-diketogulonate. This compound is further oxidized to form
oxalic acid and L- tetrose acid.
CH2OH
CH2OH
HC
O
OH
H
H
HO
HC
O
OH
ascorbic acid
OH
O
O
O
H
O
O
dehydroascorbic acid
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LAB 7: STABILITY OF INJECTIONS
Shenyang Pharmaceutical University
PHARMACEUTICS III
COOH
C
O
C
O
COOH
H
C
OH
HO
C
H
OH-
C
H
O
H2O
HO
COOH
OH
C
H
COOH
CH2OH
CH2OH
2,3-diketogulonate
L- tetrose acid
Oxalic acid
In the anaerobic conditions, dehydration and hydrolysis will occur with the
generation of furaldehyde and carbon dioxide. Because of the catalytic effect of H+,
dehydration process is faster in an acid medium than that in an alkaline medium.
CH2OH
HC
OH
O
O
-2 H2O
H
HO
- CO2
- H2O
CHO
O
+
H2O
O
O
OH
O
CO2
OH
O
C
+
COOH
H2O
Furaldehyde
The stability of a Vitamin C solution is mainly influenced by the oxygen in the air,
pH, metal ions, temperature, lighting and so on. Moisture in the product and humidity in
the environment are the two main factors affecting the stability of solid Vitamin C. The
instability of Vitamin C is indicated by the decrease in drug potency and development of a
yellow color. According to the Chinese Pharmacopoeia, the discoloration of Vitamin C
injection should be determined by spectrophotometry at a wavelength of 420 nm and the
absorption should be no more than 0.06. The iodometry method is applied to determine the
content of Vitamin C, since Vitamin C can quantitatively react with iodine due to the
reduction property of Vitamin C. The reaction scheme is shown as follows:
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LAB 7: STABILITY OF INJECTIONS
Shenyang Pharmaceutical University
CH2OH
HC
O
CH2OH
OH
O
H
HO
PHARMACEUTICS III
H+
+
HC
OH
I2
O
+
2 HI
H
OH
O
O
The purpose of this experiment is to investigate the influence of pH, oxygen in the air
and antioxidants on the stability of Vitamin C injection through monitoring the changes in
solution color and drug content.
3. PROCEDURES
3.1. Factors influencing the stability of Vitamin C injection
3.1.1 Preparation of 5% Vitamin C injection
Boil 400 mL of water for injection and cool to room temperature for use. Weigh 15 g
of Vitamin C and dissolve in the cool boiled water followed by diluting to 300 mL to make
a 5% Vitamin C solution. Determine the Vitamin C content in the solution and measure the
absorbance at 420 nm at time zero.
3.1.2 Effect of pH
The injection prepared in 3.1.1 is divided into four portions which are filled in
different containers with a volume of 30 mL, 30mL, 210mL and 30mL, respectively. A
NaHCO3 powder is used to adjust the solution pH to 4.0, 5.0, 6.0, 7.0 accordingly (the pH
is first monitored using the pH test paper and verified by using a pH meter). The solution
will be filtered using a syringe membrane filter. After filtration, the solution is filled into
2mL ampoules and 8 samples are prepared for each pH value. An additional empty
ampoule with a label (designating the pH) placed inside is tied together with the 8 solution
samples as a group. All the sample groups are placed in a 100 ℃ water bath at the same
time and heated for 1 h. Record the changes in color using “+ + +…” to indicate the
intensity of the color at different time points as listed in Table 1. At 60-min time point,
determine the drug content by recording the volume of iodine solution consumed and
measure the absorbance of the sample at 420 nm. Record all the results in Table 1.
3.1.3 The effect of oxygen in the air
100 mL of pH 6.0 Vitamin C solution prepared in part 3.1.2 is divided into three
portions: ① fill 2 mL solution into 2 mL ampoules and then seal and 8 samples are
prepared; ② fill 1 mL solution into 2 mL ampoules and then seal and 12 samples are
prepared; ③ fill 2 mL solution into 2 mL ampoules, purge with CO2 for about 5 seconds,
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Shenyang Pharmaceutical University
LAB 7: STABILITY OF INJECTIONS
PHARMACEUTICS III
sealed immediately and 8 samples are prepared. These samples are used to study the effect
of oxygen content on the stability of Vitamin C. These three group samples are placed in a
100 ℃ water bath and heated for 1 h. Record the changes in color using “+ + +…” to
designate the color intensity at different time points as listed in Table 2. At 60 min,
determine the drug content by recording the volume of iodine solution and measure the
absorbance of the injection at 420 nm. Record all the results in Table 2.
3.1.4 The effect of antioxidant
80 mL of pH 6.0 Vitamin C solution prepared in part 3.1.2 is divided into two
portions. Add 0.1 g of Na2S2O5 to one portion and use the other portion as the control. Fill
2mL in 2mL ampoules and prepare 8 samples with each portion. The samples are used to
investigate the effect of antioxidant on the stability of Vitamin C injection. These two
group samples are placed in a 100 ℃ water bath and heated for 1 h. Record the changes in
color using “+ + +…” to designate the color intensity at different time points as listed in
Table 3. At 60 min, determine the drug content by recording the volume of iodine solution
consumed and measure the absorbance of injection at 420 nm. Record all the results in
Table 3.
3.2 Assay of Vitamin C
To 1 mL of 5% Vitamin C injection (equivalent to approximately 0.05 g of Vitamin C),
add 15 mL of distilled water and 2 mL of acetone. Allow the sample to stand for 5 min
after shaking and then add 4 mL of diluted acetic acid and 1 mL of starch TS. Titrate the
solution with 0.06 mol / L iodine solution until the solution turns to blue and stay blue for
30 seconds. Record the volume (mL) of iodine solution (1 mL iodine solution is equivalent
to 5.2836 mg of Vitamin C) consumed during titration.
3.3 Notes
3.3.1 Pay special caution to safety while performing the accelerated stability test to avoid
the drying of water bath and explosion of the ampoules.
3.3.2 For the accelerated experiment, solutions in 8 samples should be combined for
determining the drug content.
3.3.3 The determination of Vitamin C content is performed using the iodometry method.
The test sample should be acidic as Vitamin C is more stable against oxidation in an
acidic medium. The samples should be titrated immediately after mixing with an
acidic medium. The presence of a reducing agent in the sample often interferes with
this determination as antioxidants such as sodium bisulfite are often added to the
formulation. In this case, acetone should be added before titration, which can react
with sulphuric acid to eliminate the interference effect of the antioxidants.
3.3.4 During the preparation of the Vitamin C solution, sodium bicarbonate should be
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LAB 7: STABILITY OF INJECTIONS
Shenyang Pharmaceutical University
PHARMACEUTICS III
added slowly to avoid the generation of a large amount of bubbles. It is also
important to stir constantly to avoid localized high alkaline pH, which may result in
the decomposition of Vitamin C.
4. RESULTS AND DISCUSSION
4.1 Record the results in the following Tables.
Table 1. Effect of pH on the stability of Vitamin C injectable solution
Change in color
Drug content
absorbance
（consumed I2 mL）
Sample
pH
10′
20′
30′
45′
60′
0′
60′
（420nm）
1
2
3
4
Conclusion
Table 2. Effect of oxygen content in the air on the stability of Vitamin C injectable solution
Drug content
Change in color
Sample
Condition
10′
20′
30′
45′
（consumed I2 mL）
60′
0′
60′
Absorbance
（420nm）
1
2
3
Conclusion
Table 3.
Influence of antioxidant on the stability of Vitamin C injectable solution
Drug content
Change in color
Sample
antioxidant
10′
20′
30′
45′
（consumed I2 mL）
60′
0′
60′
Absorbance
（420nm）
1
2
Conclusion
4.2 Discuss and analyze your results to see whether or not the results can be explained by
the theory.
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Shenyang Pharmaceutical University
LAB 7: STABILITY OF INJECTIONS
PHARMACEUTICS III
5. QUESTIONS
a) What are the main factors influencing the stability of a Vitamin C injectable
solution?
Reference
[1] Leon Lachman, Herbert A. Lieberman, Joseph L. Kanig. Theory and Practice of
Industrial Pharmacy, 3rd ed., Lea & Febiger, 1986.
[2] H. C. Ansel, N. G. Popovich and L. V. Allen, Jr. Pharmaceutical Dosage Forms and
Drug Delivery Systems, 8th ed., Williams and Wilkins, Baltimore, 2005.
(Cuifang CAI)
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