Description:FIELD OF THE INVENTION
The invention relates to a new HPLC method for determining succinyl choline chloride and related substances of the same in drug substance and in bulk of the drug substance.
BACKGROUND OF THE INVENTION
Succinyl choline chloride is chemically known as 2,2'-[(1,4-dioxo-1,4¬ butanediyl)bis(oxy)]-bis-[N,N,N-trimethylethanaminium] dichloride. Succinyl choline chloride is approved in USA for multiple uses such as general anaesthesia, to facilitate tracheal intubation, and to provide skeletal muscle relaxation during surgery or mechanical ventilation. Succinyl choline chloride absorbs UV radiation below 220 nm. Choline, a primary degradation product of succinyl choline chloride, has no detectable chromophores. Gas chromatographic methods could cause the breakdown of the ester bond of succinyl choline chloride that provides inconsistent results. Further, the determination of succinyl choline chloride and related substances of the same in the drug substance in high performance liquid chromatography (HPLC) is a challenge, due to the lack of sensitive detection methods.
In a prior art process, Succinyl choline chloride is dissolved in a mixture of glacial acetic acid and mercuric acetate and then titrated with 0.1N perchloric acid with crystal violet as indicator, wherein each ml of 0.1N perchloric acid is equivalent to 18.07 mg of succinyl choline chloride as disclosed in the Publication Analytical Profiles of Drug Substances 1981, Volume: 10, Pages: 691-704. The same publication discloses the high performance liquid chromatography (HPLC) to determine succinyl choline chloride in stainless steel column packed with Partisil with a mobile phase containing tetramethylammonium chloride in methanol adjusted pH 3 with hydrochloric acid and the UV detector was set to 214 nm.
The Publication Journal of Chromatography A 739, 1996, pages 351-357 disclose high-performance liquid chromatography (HPLC) to determine succinyl choline chloride in an Alltima C18 column 5µm, 250 X 4.6 mm (Alltech, Deerfield, IL, USA) with mobile phase containing hexane sulfonic acid as an ion-pair reagent.
The US Pharmacopoeia describes the high performance liquid chromatography (HPLC) to determine succinyl choline chloride using Cosmosil 5 µm, 4.6 x250 mm with mobile phase containing 1-pentanesulfonic acid sodium salt as an ion-pair reagent.
In existing US Pharmacopoeia method, the inventors of the present invention observed blank interference at retention time of succinyl monocholine chloride and succinic impurity and further late eluting impurity was not eluted within the runtime and elutes in the consecutive injections.
Hence, there is a need to develop a novel HPLC method for the determination of succinyl choline chloride in bulk drug.
OBJECTS OF THE INVENTION
Primary object of the invention is to provide a new HPLC method for determining succinyl choline chloride and related substances of the same in drug substance and in bulk of the drug substance.
SUMMARY OF THE INVENTION
Accordingly, the invention discloses a high pressure liquid chromatography (HPLC) method for the determination of succinyl choline chloride in bulk drug characterized in that perchloric acid is used as an ion-pair reagent in one of the two liquids A and B constituting the mobile phase. Wherein the relative concentration of the liquids A and B are varied to a gradient elution.
According to an embodiment of the invention, the liquids A and B are varied to a gradient elution is between 100% of A: 0% of B, or from 95% of A:5% of B, or from 90% of A: 10% of B or from 85% of A: 15% of B, to 100% of A: 0% of B or to 5% of A: 95% of B, or to 10% of A: 90% of B, or to 15% of A: 85% of B or to 50% of A: 50% of B; and wherein the variation in gradient elution take place over 10 to 180 minutes, preferably over 30 to 120 minutes, more preferably 30 to 70 minutes.
According to an embodiment of the invention, the liquids A and B are varied to a gradient elution is between 100% of A: 0% of B to 0% of A: 100% of B over a period of 10 to 180 minutes.
According to an embodiment of the invention, the liquids A and B are varied to a gradient elution is between preferably the gradient is between 100% of A: 0% of B to 0% of A: 100% of B over a period of 25 to 120 minutes.
According to an embodiment of the invention, the liquid A is perchloric acid solution or its mixture with water.
According to an embodiment of the invention, the liquid A is 0.5% of perchloric acid solution in water.
According to an embodiment of the invention, the liquid B is the combination of liquid A and an organic solvent.
According to an embodiment of the invention, the organic solvent is selected from acetic acid, methanol, ethanol, n-propanol, n-butanol, iso-propanol, sec-butanol, iso-butanol, tert-butanol, tetrahydrofuran, acetone, dimethoxyethane, dimethylformamide, dimethylsulfoxide, 1,4-dioxane, pyridine, acetonitrile or mixtures thereof, preferably a mixture of methanol and acetonitrile.
According to an embodiment of the invention, the liquid B comprises 5 to 90% v/v, preferably 30 to 80% v/v, more preferably 15 to 60% v/v of the organic solvent.
According to an embodiment of the invention, the mobile phase flow rate is between 0.01 and 10 ml/min, more preferably between 0.1 and 4 ml/min, most preferably 1 ml/min.
According to an embodiment of the invention, the stationary phase is selected from octadecylsilyl silica gel, octylsilyl silica gel, phenylalkyl silica gel, cyanopropyl silica gel, alkyl-diol silica gel, preferably YMC triart PFP (250 X 4.6) mm, 5µm column.
According to an embodiment of the invention, the stationary phase has a pore size of between 10 and 1000 A, or between 20 and 400 A, or between 50 to 150 A, more preferably the stationary phase a pore size of about 120°A.
According to an embodiment of the invention, the process is carried out at a column temperature between 10 to 150℃, preferably 15 to 60℃.
According to an embodiment of the invention, the eluent is analyzed by UV or visible spectrophotometer as a detector, preferably in the range of 200 to 218 nm, more preferably 214 nm.
FIGURE ACCOMPANYING THE DESCRIPTION
Figure-1: HPLC Chromatogram obtained by performing the procedures as in Example-1

DETAILED DESCRIPTION OF THE INVENTION
One embodiment of the present invention is to provide a HPLC method for determining the succinyl choline chloride and related substances of succinyl choline chloride in drug substance, wherein perchloric acid is used as an ion-pair reagent in the mobile phase, comprising
(i) a mobile phase consisting of liquid A and liquid B, wherein the relative concentration of the liquid A and B is varied to a predetermined gradient elution;
(ii) a stationary phase which is selected from octadecylsilyl silica gel, octylsilyl silica gel, phenylalkyl silica gel, cyanopropyl silica gel, alkyl-diol silica gel; and
(iii) a UV detector.
The liquid A according to the present invention is perchloric acid solution or its mixture with water. The perchloric acid solution or its mixture with water is the buffer solution employed in the HPLC method of the present invention.
The concentration of perchloric acid in the buffer solution is at a concentration of 0.001 to 0.1M, preferably at a concentration 0.001 to 0.05M.
Preferably, the liquid is 0.5% of perchloric acid solution in water (Liquid A).
The liquid B is liquid A with one or more organic solvents thereof. The organic solvent is selected from acetic acid, methanol, ethanol, n-propanol, n-butanol, iso-propanol, sec-butanol, iso-butanol, tert-butanol, tetrahydrofuran, acetone, dimethoxyethane, dimethylformamide, dimethylsulfoxide, 1,4-dioxane, pyridine, acetonitrile or mixtures thereof, preferably a mixture of methanol and acetonitrile.
In another embodiment, the liquid B comprises 5 to 90% v/v, preferably 30 to 80% v/v, more preferably 15 to 60% v/v of the organic solvent.
The method of first aspect of the present invention may comprise a gradient programming so that the relative concentration of the liquids A and B are varied to a gradient is between 100% of A: 0% of B to 0% of A: 100% of B over a period of 10 to 180 minutes. Preferably the gradient is between 100% of A: 0% of B to 0% of A: 100% of B over a period of 25 to 120 minutes.
All used herein in relation to any aspect of the present invention, unless stated otherwise all percentages given in relation to the concentration of liquids A and/or B refer to the percentage by volume.
Alternatively, the first aspect of the present invention comprise a gradient programming so that the relative concentration of the liquids A and B is varied to a gradient from about 100% of A: 0% of B, or from about 95% of A:5% of B, or from about 90% of A: 10% of B or from about 85% of A: 15% of B, to about 100% of A: 0% of B or to about 5% of A: 95% of B, or to about 10% of A: 90% of B, or to about 15% of A: 85% of B or to about 50% of A: 50% of B. The variation in gradient may take place over 10 to 180 minutes, preferably over 30 to 120 minutes, more preferably 30 to 70 minutes.
A particularly preferred method according to one aspect of the present invention is when the liquid A is 0.5% of perchloric acid in water (buffer) and the liquid B is a mixture of liquid A, methanol and acetonitrile in the ratio of 1:1 v/v and the gradient is as follows.
Time (Minutes) Liquid A (in %) Liquid B (in %)
0.01 98 2
10 98 2
20 85 15
25 80 20
45 50 50
47 98 2
60 98 2

Preferably the mobile phase flow rate between 0.01 and 10 ml/min is used, more preferably a mobile phase flow rate between 0.1 and 4 ml/min is used, most preferably 1 ml/min is used.
Typically, the method of the first aspect of the current invention is carried out at a column temperature between 10 to 150℃, preferably 15 to 60℃.
In one embodiment of the present invention the stationary phase is selected from octadecylsilyl silica gel, octylsilyl silica gel, phenylalkyl silica gel, cyanopropyl silica gel, alkyl-diol silica gel, preferably YMC triart PFP (250 X 4.6) mm, 5µm column.
Preferably the stationary phase has a particle size of between 0.1 and 100 µm or between 0.5 and 25 µm, or between 1 and 10 µm, more preferably has a particle size of about 5 µm.
Preferably the stationary phase has a pore size of between 10 and 1000 A, or between 20 and 400 A, or between 50 to 150 A, more preferably the stationary phase a pore size of about 120°A.
In an embodiment of the invention, the chromatography is carried out in a column between 10 mm and 500 mm in length, or in a column between 300 mm and 400 mm in length or between 250 mm and 500 mm in length, preferably the chromatography is carried out in a column about 250 mm in length.
The chromatography may be carried out in a column between 0.01 mm and 25 mm in internal diameter, or between 0.1 mm and 30 mm in internal diameter, or between 1mm and 10mm in internal diameter, preferably the chromatography is carried out in a column about 4.6 mm in internal diameter.
The eluent is analyzed by UV or visible spectrophotometer as a detector, preferably in the range of 200 to 218 nm, more preferably 214 nm.
The HPLC method of the present invention quantifies succinyl choline chloride in the drug substance both in small and bulk quantities and is also used to determine the percentage of the succinyl choline chloride in bulk drug batches.
In another embodiment of the present invention, HPLC method for analyzing related substances of succinyl choline chloride having chromatographic conditions provided in the below table.
Liquid A 0.5% Perchloric acid in water (Buffer solution)
Liquid B Mixture of Liquid A with methanol and acetonitrile (20: 40: 40)
Stationary phase YMC triart PFP (250 X 4.6) mm, 5µm column
Wavelength 214 nm
Flow rate 1.0 ml/ minutes
Sample concentration 10 mg/ ml
Column oven temperature 20
Column cooler temperature 10
Injection volume 50 µl
Run time 60.01 minutes
Elution mode Gradient

The present invention is explained in detail with reference to the following examples described below, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.

Example
Experimental conditions
Stationary phase: YMC triart PFP (250 X 4.6) mm, 5µm column
Flow rate:
Detection: Sample concentration: Diluent
Liquid A: 0.5% Perchloric acid in water (Buffer solution)
Liquid B: Mixture of Liquid A with methanol and acetonitrile in the ratio 20: 40: 40
Mobile phase: Liquid A- Liquid B gradient
Time (Minutes) Liquid A (in %) Liquid B (in %)
0.01 98 2
10 98 2
20 85 15
25 80 20
45 50 50
47 98 2
60 98 2

 
, Claims:1. A high-pressure liquid chromatography (HPLC) method for the determination of succinyl choline chloride in bulk drug characterized in that perchloric acid is used as an ion-pair reagent in one of the two liquids A and B constituting the mobile phase.

2. The high-pressure liquid chromatography method for the determination of succinyl choline chloride in bulk drug as claimed in claim 1 wherein the relative concentration of the liquids A and B are varied to a gradient elution.

3. The high-pressure liquid chromatography method for the determination of succinyl choline chloride in bulk drug as claimed in claim 2 wherein the liquids A and B are varied to a gradient elution is between 100% of A: 0% of B, or from 95% of A:5% of B, or from 90% of A: 10% of B or from 85% of A: 15% of B, to 100% of A: 0% of B or to 5% of A: 95% of B, or to 10% of A: 90% of B, or to 15% of A: 85% of B or to 50% of A: 50% of B; and wherein the variation in gradient elution take place over 10 to 180 minutes, preferably over 30 to 120 minutes, more preferably 30 to 70 minutes.

4. The high-pressure liquid chromatography method for the determination of succinyl choline chloride in bulk drug as claimed in claim 3 wherein the liquids A and B are varied to a gradient elution is between 100% of A: 0% of B to 0% of A: 100% of B over a period of 10 to 180 minutes.

5. The high-pressure liquid chromatography method for the determination of succinyl choline chloride in bulk drug as claimed in claim 3 wherein the liquids A and B are varied to a gradient elution is between preferably the gradient is between 100% of A: 0% of B to 0% of A: 100% of B over a period of 25 to 120 minutes.

6. The high-pressure liquid chromatography method for the determination of succinyl choline chloride in bulk drug as claimed in claim 1 wherein the liquid A is perchloric acid solution or its mixture with water.

7. The high-pressure liquid chromatography method for the determination of succinyl choline chloride in bulk drug as claimed in claim 6 wherein the liquid A is 0.5% of perchloric acid solution in water.

8. The high-pressure liquid chromatography method for the determination of succinyl choline chloride in bulk drug as claimed in claim 1 wherein the liquid B is the combination of liquid A and an organic solvent.

9. The high-pressure liquid chromatography method for the determination of succinyl choline chloride in bulk drug as claimed in claim 8 wherein the organic solvent is selected from acetic acid, methanol, ethanol, n-propanol, n-butanol, iso-propanol, sec-butanol, iso-butanol, tert-butanol, tetrahydrofuran, acetone, dimethoxyethane, dimethylformamide, dimethylsulfoxide, 1,4-dioxane, pyridine, acetonitrile or mixtures thereof, preferably a mixture of methanol and acetonitrile.

10. The high-pressure liquid chromatography method for the determination of succinyl choline chloride in bulk drug as claimed in claim 8 wherein the the liquid B comprises 5 to 90% v/v, preferably 30 to 80% v/v, more preferably 15 to 60% v/v of the organic solvent.

11. The high-pressure liquid chromatography method for the determination of succinyl choline chloride in bulk drug as claimed in claim 1 wherein the mobile phase flow rate is between 0.01 and 10 ml/min, more preferably between 0.1 and 4 ml/min, most preferably 1 ml/min.

12. The high-pressure liquid chromatography method for the determination of succinyl choline chloride in bulk drug as claimed in claim 1 wherein the stationary phase is selected from octadecylsilyl silica gel, octylsilyl silica gel, phenylalkyl silica gel, cyanopropyl silica gel, alkyl-diol silica gel, preferably YMC triart PFP (250 X 4.6) mm, 5µm column.

13. The high-pressure liquid chromatography method for the determination of succinyl choline chloride in bulk drug as claimed in claim 12 wherein the stationary phase has a pore size of between 10 and 1000 A, or between 20 and 400 A, or between 50 to 150 A, more preferably the stationary phase a pore size of about 120°A.

14. The high-pressure liquid chromatography method for the determination of succinyl choline chloride in bulk drug as claimed in claim 1 wherein said process is carried out at a column temperature between 10 to 150℃, preferably 15 to 60℃.

15. The high-pressure liquid chromatography method for the determination of succinyl choline chloride in bulk drug as claimed in claim 1 wherein the eluent is analyzed by UV or visible spectrophotometer as a detector, preferably in the range of 200 to 218 nm, more preferably 214 nm.