DESC:Field of Invention:
The present invention relates to a composition and method for the preparation of a stable aqueous solution comprising combination of ibuprofen and paracetamol or pharmaceutically acceptable salts thereof for intravenous injection or infusion.
Background of the invention:
The combination of Paracetamol and Ibuprofen is used to provide relief from mild to moderate pain including postoperative pain, dysmenorrhoea and musculoskeletal pain and for reduction of fever. Although the combination is available in oral dosage form but in case of emergency where patients required rapid onset of action or unable to swallow or if have postoperative nausea and vomiting, or reduced gastric motility, oral dosage form have certain limitations.
MAXIGESIC® IV by AFT Pharma is a marketed intravenous(IV) infusion solution which is directly administered to the vein and is used to reduce fever and relieve pain. The composition is described in patent number US11213498B2 which discloses use of acetyl cysteine or cysteine to improve the compatibility and stability as Paracetamol is known to undergo hydrolysis in aqueous solution. The formation of p-aminophenol following hydrolysis of paracetamol which itself can degrade into quinoneimine is for described in “Acetaminophen” in Analytical Profiles of Drug Substances, 1974, vol. 3.
However, the patent further discloses that Acetyl cysteine or cysteine causes yellow coloration and the inventors of the present study have further found that if the solution containing cysteine is heat sterilized especially at high temperatures in the region of 120° C, the solution show brownish color after storage.
This issue was also cited in Patent US8404891 B2 which further disclosed that cysteine hydrochloride is degraded at high temperature and due to heat action during sterilization, a dimer of paracetamol may be formed as a degradation product and which increases during storage with other unknown degradation products. To avoid the dimer formation and cysteine degradation the prior art used additional process steps i.e. admixing of the paracetamol to the aqueous solvent that has a temperature of between 65° C. and 95° C., cooling the solution so formed to a temperature below 40° C., preferably equal to or above 35° C. and below 40° C., and then adding cysteine hydrochloride and NaOH and it was observed that combining above mentioned steps i.e. temperature, cooling and cysteine, dimer formation was avoided during sterilization, and increase of its level during storage was also avoided. However, the prior art required additional process steps and therefore required considerable amount of time.
Depending on the technique used for the removal of oxygen from paracetamol injection a considerable amount of time is required. Another disadvantage is that oxidation products lead to the formation of colored compounds, making the aqueous solution unsuitable for therapeutic applications. The problem becomes more complicated when the intravenous infusion is designed to contain both paracetamol and ibuprofen.
To avoid the instability of Cysteine during heat sterilization and coloration during storage, the inventors of the present invention also studied the use of Sodium metabisulfate as antioxidants. The use of Sodium metabisulfate as an antioxidant in the preparation of Paracetamol infusion is already known in the art. WO2011/071400 discloses a method to prepare liquid solution of paracetamol which contains both cysteine and sodium metabisulphite and further required a deoxygenation of the head space of the storage tanks with inert gas. This makes the process complicated and further there was no disclosure and the stability if another active ingredient like Ibuprofen was added to the solution. IN2202/MUM/2009 also teaches the use of sodium metabisulfite in combination is removal of oxygen by purging the solution with a water insoluble inert gas. But the prior art didn’t teach on how to prepare a stable formulation if Ibuprofen was added to the same.
The inventors of the present invention surprisingly found that when a stabilizing/solubilizing agent comprising is added to sodium metabisulfate a stable formulation of Ibuprofen and paracetamol can be obtained. The use of Sodium metabisulfate together with solubilizing/stabilizing agent also avoids the additional steps which was required to avoid degradation of Cysteine and dimer formation.
Summary
The current invention provides a solution for at least one of the above mentioned problems by providing a composition combining paracetamol and ibuprofen with improved compatibility and reduced tendency to undergo hydrolysis and oxidation.
The present invention provides a stable composition of an aqueous solution comprising combination of ibuprofen and paracetamol or pharmaceutically acceptable salts thereof with one more anti-oxidants other than Cysteine or Acetyl cysteine.
In another aspect the present invention provides a simple process for manufacturing of an aqueous composition which provide a stable formulation which prevent the physical discoloration and avoid dimer formation of paracetamol which was associated with usage of cysteine including its salts, isomers or other derivatives.
In yet another aspect the present invention provides a parenteral formulation comprising:
a) Paracetamol or its pharmaceutically acceptable salts present at a concentration ranging from 8-12 mg per ml and;
b) Ibuprofen or its pharmaceutically acceptable salts present at a concentration ranging from 2-4 mg per ml and;
c) Sodium metabisulphite as antioxidant which prevents the degradation of paracetamol and provide over all stability to the aqueous solution
d) L-arginine as solubilizing or stabilizing agent.
Wherein parenteral formulation is in form of intravenous injection or infusion.
In another embodiment the present invention provides an aqueous solution of Paracetamol and Ibuprofen or pharmaceutically acceptable salts thereof which when stored in infusion container remains stable even after thermal sterilization at 121°C for 20 min.
In yet another aspect the present invention provides a simple manufacturing process of an aqueous composition comprising ibuprofen and paracetamol in combination which doesn’t not required additional process steps which were earlier required to prevent the degradation of Cysteine and dimer formation of paracetamol. The process includes following steps:
a. Purge the water for injection (80oC -85oC) with nitrogen gas throughput the manufacturing process and cool down the water for injection till 75oC -80oC and;
b. Dissolve L-arginine into step a and stir the bulk solution at 300 RPM for 10 min and Cool down the solution till 60oC -65oC and;
c. Add & dissolve Ibuprofen into the bulk solution obtained in step b and under constant stirring at 300 RPM for 20 minutes. Cool down the solution till 40°C -45°C.
d. Add & dissolve Mannitol into step c and stir the bulk solution at 300 RPM for 10 min and Cool down the solution till 35oC -40oC.
e. Add & dissolve Sodium Metabisulphite into step d and stir the bulk solution at 300 RPM for 10 min. Cool down the solution till 30oC -35oC.
f. Add & dissolve Paracetamol into the bulk solution step-e under constant stirring at 300 RPM for 20 min.
g. Check the pH of bulk solution (6.50 to 7.00); If required, adjust the pH between 6.50 to 7.00 by using 10% v/v Glacial Acetic acid Solution.
Detailed Description of invention:
Before the present materials and methods are described, it is understood that this invention is not limited to the particular methodology, protocols, materials, and reagents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only.
It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. As well, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising", "including", and "having" can be used interchangeably.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications and patents specifically mentioned herein are incorporated by reference for the purposes of describing and disclosing the chemicals, instruments, statistical analysis and methodologies which are reported in the publications which might be used in connection with the invention. All references cited in this specification are to be taken as indicative of the level of skill in the art. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
The term “effective amount” as used in the present invention, refers to an amount of active ingredients that provides the desired pharmacological effects when administered according to a dose appropriate for each administration route.
The term “pharmaceutically acceptable excipients” as used in the present invention, refers to ingredients other than active ingredients which compatible with other ingredients, and that are not harmful to human beings or animals and does not have any pharmacological activities.
The term “antioxidants” as used in the present invention, refers to the compounds that decrease or prevent the oxidation of Paracetamol and provide stability of overall composition.
The term “infusion” as used in the present invention refers to the dosage form which is directly administered into a vein via catheter.
The present invention provides a stable intravenously administrable composition comprising an effective amount of Paracetamol and Ibuprofen or pharmaceutically effective salts thereof wherein intravenous administration is in form of infusion.
In an embodiment the present invention provides a stable liquid formulation comprising an effective amount of Paracetamol and Ibuprofen in an aqueous solvent.
In an embodiment paracetamol in composition is in the form of a free base.
In still other embodiment the paracetamol present in composition is present in range of 5-15 mg/mL more preferably 10 mg/ml equivalent to 1.0 % w/v.
In an another embodiment ibuprofen in composition is present in form of salt. e.g. sodium salt or may be present in base form.
In some embodiment ibuprofen or pharmaceutically acceptable salts thereof and is present in range of 1-5 mg/ml more preferably 3 mg/ml equivalent to 0.3%w/v.
In another embodiment the present invention provides an intravenously administrable composition comprising 10 mg/ml paracetamol and 3 mg/ml ibuprofen in an aqueous carrier form.
In an another embodiment the present invention provides pharmaceutical formulation which is cysteine free.
In an embodiment the present invention provides an intravenous infusion dosage form comprising combination of effective amount of paracetamol and ibuprofen for treatment of pain or reduction of fever which is administered to the patients in case of emergency where patients are unable to swallow tablet or capsule and required rapid onset of action or in state of unconsciousness or patient suffering from nausea and vomiting or other gastro-intestinal disorders.
In another embodiment the present invention provides an intravenous infusion dosage form comprising:
i. Paracetamol or its pharmaceutically acceptable salts present at a concentration ranging from 8-12 mg per ml and;
ii. Ibuprofen or its pharmaceutically acceptable salts present at a concentration ranging from 2-4 mg per ml and;
iii. Sodium metabisulphite as antioxidant which prevents the degradation of paracetamol and provide over all stability to the aqueous solution
iv. L-arginine as solubilizing or stabilizing agents
Wherein Sodium metabisulphite is present in an amount 0.01% to 0.05% of the total volume of the solution more preferably 0.02% w/v.
In another embodiment the present invention provides an aqueous solution of Paracetamol and Ibuprofen or pharmaceutically acceptable salts thereof which when stored in infusion container remains stable even after thermal sterilization at 121°C for 20 min.
The pH of the compositions of the present invention can be modified by utilizing a sufficient amount of a pH adjuster selected from the group consisting of an acid and a base. Suitable pH adjusters typically include at least an acid or a salt thereof, and/or a base or a salt thereof.
In a preferred embodiment pH adjuster is selected form but not limited to group consisting of hydrochloric acid, phosphoric acid, citric acid, ascorbic acid, acetic acid, sulphuric acid, carbonic acid and nitric acid, sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate and magnesium hydroxide more preferably combination of acetic acid and sodium hydroxide.
In another preferred embodiment, the said aqueous solution has a pH of 5 to 8..
In an another embodiment the aqueous solution of present invention further comprises tonicity agent selected from group consisting of anhydrous or hydrous forms of sodium chloride, dextrose, sucrose, xylitol, fructose, glycerol, sorbitol, mannitol, potassium chloride, mannose, calcium chloride, magnesium chloride and other inorganic salts. Preferably, the tonicity agent is Mannitol.
In an another embodiment tonicity agent can be utilized in the formulations of the present invention in an amount 2-10% w/v more preferably 2.5%w/v.
In an another embodiment the intravenous formulation may optionally further comprises pharmaceutically acceptable buffer. Suitable buffers include, but are not limited to, pharmaceutically acceptable salts and acids of acetate, glutamate, citrate, tartrate, benzoate, lactate, histidine or other amino acids, gluconate, phosphate, malate, succinate, formate, propionate, and carbonate.
In another embodiment the present invention comprises one or more solubilizer selected from lactic acid, arginine, ascorbic acid, acetic acid, propionic acid, succinic acid, gluconic acid, benzoic acid, tartaric acid, glutaric acid, malic acid, and fumaric acid.
In another embodiment the preferred solubilizer is arginine present in an amount ranging from 0.05 to 2% more preferably 0.4%.
In another embodiment the present invention provides a stable, colorless and clear intravenous infusion formulation.
In an embodiment the present invention provides intravenous infusion comprising:
i. 0.5 to 5.0 % w/v of paracetamol or pharmaceutically acceptable salts thereof and;
ii. 0.05 to 1% w/v of Ibuprofen or pharmaceutically acceptable salts thereof and;
iii. 2-10% w/v of tonicity agent and;
iv. 0.01% to 0.5% w/v of antioxidant and;
v. 0.05% to 2% w/v of solubilizer
The present invention provides an intravenous formulation comprising following:
i. 1.0 % w/v paracetamol or pharmaceutically acceptable salts thereof and;
ii. 0.3 % w/v ibuprofen or pharmaceutically acceptable salts thereof and;
iii. 2-10% w/v mannitol as tonicity agent and;
iv. 0.01% to 0.5% w/v of sodium metabisulphite as antioxidant and
v. 0.05% to 2% w/v of L-arginine as solubilizer
One or more pH adjusting agents and wherein the water is used as a vehicle.
In another embodiment the present invention provides sterile infusion solution comprising:
i. 1% w/v of Paracetamol and;
ii. 0.3 % w/v of Ibuprofen sodium and;
iii. 3.2% w/v of mannitol as tonicity agent and;
iv. 0.05% w/v of sodium metabisulphite as antioxidant and;
v. 0.4% w/v of L-Arginine as solubilizer
In another embodiment the present invention provides process of preparing infusion formulation comprising following steps:
a. Purge the water for injection (80oC -85oC) with nitrogen gas throughput the manufacturing process and cool down the water for injection till 75oC -80oC and;
b. Dissolve L-arginine into step a and stir the bulk solution at 300 RPM for 10 min and Cool down the solution till 60oC -65oC and;
c. Add & dissolve Ibuprofen into the bulk solution obtained in step b and under constant stirring at 300 RPM for 20 minutes. Cool down the solution till 40°C -45°C.
d. Add & dissolve Mannitol into step c and stir the bulk solution at 300 RPM for 10 min and Cool down the solution till 35oC -40oC.
e. Add & dissolve Sodium Metabisulphite into step d and stir the bulk solution at 300 RPM for 10 min. Cool down the solution till 30oC -35oC.
f. Add & dissolve Paracetamol into the bulk solution step-e under constant stirring at 300 RPM for 20 min.
g. Check the pH of bulk solution (6.50 to 7.00); If required, adjust the pH between 6.50 to 7.00 by using 10% v/v Glacial Acetic acid Solution.
EXAMPLES
To illustrate non-limiting embodiments of the present disclosure, the following examples were prepared.
Example-1
Table-1: FORMULATION-A
Ingredients Concentration
Paracetamol 1.00%
Ibuprofen sodium eq to ibuprofen 0.30%
Sodium chloride 0.90%
Sodium citrate 0.30%
Sodium acetate 0.30%
Acetic acid q.s. to pH 6.3
Water for injection q.s

Example-2
Table 2- FORMULATION-B
Ingredients Concentration
Paracetamol 1.00%
Ibuprofen sodium eq to ibuprofen 0.30%
Mannitol 3.29%
Sodium citrate 0.30%
Sodium metabisulphite 0.02%
Sodium acetate 0.30%
Acetic acid q.s. to pH 6.25
Water for injection q.s

Example-3
Table 3- FORMULATION-C
Ingredients Concentration
Paracetamol 1.00%
Ibuprofen sodium eq to ibuprofen 0.30%
Mannitol 3.29%
Sodium citrate 0.30%
Sodium metabisulphite 0.01%
Sodium acetate 0.30%
Acetic acid q.s. to pH 6.31
Water for injection q.s

Example-4
Table 4- FORMULATION-D
Ingredients Concentration
Paracetamol 1.00%
Ibuprofen sodium eq to ibuprofen 0.30%
Mannitol 3.29%
Sodium citrate 0.30%
Sodium metabisulphite 0.01%
Sodium acetate 0.30%
Acetic acid q.s. to pH 6.31
Water for injection q.s

Example-5
Table 5- Formulation-E
Ingredients Concentration
Paracetamol 1.00%
Ibuprofen sodium dihydrate eq to ibuprofen 0.30%
Mannitol 3.2%
L-Arginine 0.4%
Sodium meta bisulphite 0.05%
Acetic acid (Glacial) q.s.
Water for Injections q.s.

Collect approximately 2000 Ltr. Water for Injections IP (80°C -85°C) in 2500 Ltr. Batch size, in a compounding vessel and purge 0.2 µ filtered nitrogen gas throughout manufacturing process. Check the dissolved oxygen content in collected Water for Injections IP (DO Limit: NMT 2 mg/Litre). Cool down the WFI till 75°C -80°C. Dissolve L-Arginine in water for injection and stir the solution at 300 RPM for 10 min. Cool down the solution till 60 -65. To this solution add Ibuprofen with constant stirring at 300 RPM for 20 minutes. Cool down the solution till 40oC -45oC. To this solution add mannitol and stir the bulk solution at 300 RPM for 10 min. Cool down the solution till 35oC -40oC. To the obtain solution add & dissolve Sodium Metabisulphite and stir the bulk solution at 300 RPM for 10 min. Cool down the solution till 30oC -35oC. Add & dissolve Paracetamol to above solution under constant stirring at 300 RPM for 20 min. Check the pH of bulk solution (6.50 to 7.00); If required, adjust the pH between 6.50 to 7.00 by using 10% v/v Glacial Acetic acid Solution.
Make up the final volume to up to 2500 Liters with Water for Injections IP (25°C-30°C) and stirring the solution for 15 min at 300 RPM.
Example-6 Stability study
Example 6(a)- The paracetamol and Ibuprofen solution for infusion of example-5 was subjected to stability study under controlled room temperature (Temp. 25°C ± 2°C)
Test Acceptance criteria Initial testing After 07 days After 14 days After 21 days After 28 days
Description Clear colourless liquid filled in clear transparent glass vial Clear colourless liquid filled in clear transparent glass vial Clear colourless liquid filled in clear transparent glass vial Clear colourless liquid filled in clear transparent glass vial Clear colourless liquid filled in clear transparent glass vial Clear colourless liquid filled in clear transparent glass vial
pH Between 6.30 to 7.30 6.72 6.61 6.62 6.60 6.63
Related substance (by HPLC) As below
Ibuprofen As below
Ibuprofen Impurity J Not more than 0.3% 0.051% 0.046 0.041 0.042 0.041
Ibuprofen Impurity A Not more than 0.3% Not detected Not detected Not detected Not detected Not detected
Ibuprofen Impurity B Not more than 0.3% Not detected Not detected Not detected Not detected Not detected
Single Maximum impurity Not more than 0.3% 0.024% Not detected Not detected Not detected Not detected
Total impurities Not more than 2.0 % 0.084% 0.046% 0.041% 0.042% 0.041%
Paracetamol As below
4-Aminophenol impurity Not more than 0.1% Not detected Not detected Not detected Not detected Not detected
4-chloroacetanilide
impurity Not more than 10 ppm Not detected Not detected Not detected Not detected Not detected
Single maximum impurity Not nore than 0.25% 0.004% 0.005% 0.004% 0.004% 0.005%
Total impurities Not more than 1.0% 0.010% 0.014% 0.014% 0.013% 0.013%
Assay (by HPLC)
Each 100 ml contains
Paracetamol IP
1000 mg Not less than 900.00mg & not more than 1100.00mg
(not less than 90.00% & not more than 110.00% of label claimed) 1023.00 mg
102.30% 1026.22 mg
102.62% 1006.23 mg
100.62% 1011.91 mg
101.19% 1008.83 mg
100.88%
Ibuprofen IP
300 mg
(As sodium dihydrate) Not less than 270.00mg & not more than 330.00mg
(not less than 90.00% & not more than 110.00% of label claimed) 298.00 mg
99.33% 295.25 mg
98.14% 293.71 mg
97.90% 294.20 mg
98.07% 293.18 mg
97.73%

CONCLUSION: The infusion solution prepared from example 5 was found to be stable after 28 days of controlled room temperature (Temp. 25°C ± 2°C)
Example 6(b): The paracetamol and Ibuprofen solution for infusion of example-5 was subjected to stability study under temperature 50°C ± 2°C
Test Acceptance criteria Initial testing After 07 days After 14 days After 21 days After 28 days
Description Clear colourless liquid filled in clear transparent glass vial Clear colourless liquid filled in clear transparent glass vial Clear colourless liquid filled in clear transparent glass vial Clear colourless liquid filled in clear transparent glass vial Clear colourless liquid filled in clear transparent glass vial Clear colourless liquid filled in clear transparent glass vial
pH Between 6.30 to 7.30 6.72 6.71 6.74 6.75 6.74
Related substance (by HPLC) As below
Ibuprofen As below
Ibuprofen Impurity J Not more than 0.3% 0.051% 0.04% 0.044% 0.041% 0.04%
Ibuprofen Impurity A Not more than 0.3% Not detected Not detected Not detected Not detected Not detected
Ibuprofen Impurity B Not more than 0.3% Not detected Not detected Not detected Not detected Not detected
Single Maximum impurity Not more than 0.3% 0.024% Not detected Not detected Not detected Not detected
Total impurities Not more than 2.0 % 0.084% 0.04% Not detected Not detected Not detected
Paracetamol As below
4-Aminophenol impurity Not more than 0.1% Not detected 0.04% Not detected Not detected Not detected
4-chloroacetanilideimpurity Not more than 10 ppm Not detected Not detected Not detected Not detected Not detected
Single maximum impurity Not nore than 0.25% 0.004% 0.039% 0.033% 0.036% 0.220%
Total impurities Not more than 1.0% 0.010% 0.052% 0.046% 0.05% 0.265%
Assay (by HPLC): Each 100 ml contains
Paracetamol IP
1000 mg Not less than 900.00mg & not more than 1100.00mg
(not less than 90.00% & not more than 110.00% of label claimed) 1023.00 mg
102.30% 996.49 mg
99.65% 996.56 mg
99.66% 998.86 mg
99.89% 1005.47 mg
100.55%
Ibuprofen IP
300 mg
(As sodium dihydrate) Not less than 270.00mg & not more than 330.00mg
(not less than 90.00% & not more than 110.00% of label claimed) 298.00 mg
99.33% 289.81mg
96.60% 289.86 mg
96.62% 290.61 mg
96.87% 290.04 mg
96.68%

The paracetamol and ibuprofen intravenous solution was found to be stable after 28 days when stored at temperature 50°C ± 2°C.
,CLAIMS:1. A stable intravenously administrable composition comprising an effective amount of Paracetamol and Ibuprofen or pharmaceutically effective salts thereof wherein intravenous administration is in form of infusion.
2. The stable intravenously administrable composition as claimed in claim 1 wherein composition is free from cysteine including its salts, isomers or other derivatives.
3. The stable intravenously administrable composition as claimed in claim 1 is a colorless and clear intravenous infusion formulation.
4. The stable intravenous infusion formulation as claimed in previous claims provides an aqueous solution of Paracetamol and Ibuprofen or pharmaceutically acceptable salts thereof which when stored in infusion container remains stable even after thermal sterilization at 121°C for 20 min.
5. The stable intravenous infusion as claimed in claim 4 comprises:
i. 0.5 to 5.0 % w/v of paracetamol or pharmaceutically acceptable salts thereof and;
ii. 0.05 to 1% w/v of Ibuprofen or pharmaceutically acceptable salts thereof and;
iii. 2-10% w/v of tonicity agent and;
iv. 0.01% to 0.5% w/v of antioxidant and;
v. 0.05% to 2% w/v of solubilizer
6. The stable intravenous infusion as claimed in claim 5 further comprises:
i. 1% w/v of Paracetamol and;
ii. 0.3 % w/v of Ibuprofen sodium and;
iii. 3.2% w/v of mannitol as tonicity agent and;
iv. 0.05% w/v of sodium metabisulphite as antioxidant and;
v. 0.4% w/v of L-Arginine as solubilizer
wherein composition is free from cysteine including its salts, isomers or other derivatives.
7. The process of preparing intravenous infusion as claimed in claim 1-6 comprises following steps:
a. Purge the water for injection (80°C -85°C) with nitrogen gas throughput the manufacturing process and cool down the water for injection till 75°C -80°C and;
b. Dissolve L-arginine into step a and stir the bulk solution at 300 RPM for 10 min and Cool down the solution till 60°C -65°C and;
c. Add & dissolve Ibuprofen into the bulk solution obtained in step b and under constant stirring at 300 RPM for 20 minutes. Cool down the solution till 40°C -45°C.
d. Add & dissolve Mannitol into step c and stir the bulk solution at 300 RPM for 10 min and Cool down the solution till 35°C -40°C.
e. Add & dissolve Sodium Metabisulphite into step d and stir the bulk solution at 300 RPM for 10 min. Cool down the solution till 30°C -35°C.
f. Add & dissolve Paracetamol into the bulk solution step-e under constant stirring at 300 RPM for 20 min.
g. Check the pH of bulk solution (6.50 to 7.00); If required, adjust the pH between 6.50 to 7.00 by using 10% v/v Glacial Acetic acid Solution.