Claims:
1) An analytical method to detect and quantitating melamine in a sample of Active Pharmaceutical Ingredients (APIs) comprises a) subjecting the sample to chromatography on to a column of C18, (250mm × 4.6 mm, 5 µm) b) eluting the sample with mobile phase A and B and c) determining the quantity of melamine in the sample; wherein mobile phase A is aqueous solution comprising tetrabutyl ammonium hydroxide and mobile phase B is Acetonitrile.
2) The analytical method according to claim 1, wherein the concentration of tetrabutyl ammonium hydroxide aqueous solution is 0.08 to 0.12% V/V.
3) The analytical method according to claim 2, wherein the concentration of tetrabutyl ammonium hydroxide aqueous solution is 0.10% V/V.
4) The analytical method according to claim 1, wherein the column C18, (250mm × 4.6 mm, 5 µm) is having more than 16% carbon load.
5) The analytical method according to claim 1, wherein the column C18, (250mm × 4.6 mm, 5 µm) is Unisphere Extend C18, (250mm × 4.6 mm, 5 µm).
6) The analytical method according to claim 1, wherein the mobile phase A and mobile phase B is used with the following gradient program wherein the proportions of the mobile phases are all volume percentages as mentioned below.
Time (min.) Flow (ml/min) Mobile Phase A (%) Mobile Phase B (%)
0.0 1.0 100.0 0.0
10.0 1.0 100.0 0.0
15.0 1.0 20.0 80.0
25.0 1.0 20.0 80.0
30.0 1.0 100.0 0.0
50.0 1.0 100.0 0.0

7) An analytical system for determination and quantification of melamine in Active Pharmaceutical Ingredients (APIs) comprises a Unisphere Extend C18, (250mm × 4.6 mm, 5 µm) column and mobile phase A and B wherein mobile phase A is aqueous solution comprising tetrabutyl ammonium hydroxide and mobile phase B is Acetonitrile.
8) The analytical system according to claim 7, wherein the column C18, (250mm × 4.6 mm, 5 µm) is having more than 16% carbon load.
9) The analytical system according to claim 7, wherein, the concentration of tetrabutyl ammonium hydroxide aqueous solution is 0.08 to 0.12% V/V.
10) The analytical system according to claim 7, wherein the mobile phase A and mobile phase B is used with the following gradient program wherein the proportions of the mobile phases are all volume percentages as mentioned below.
Time (min.) Flow (ml/min) Mobile Phase A (%) Mobile Phase B (%)
0.0 1.0 100.0 0.0
10.0 1.0 100.0 0.0
15.0 1.0 20.0 80.0
25.0 1.0 20.0 80.0
30.0 1.0 100.0 0.0
50.0 1.0 100.0 0.0
, Description:Field of the invention:
The present invention relates to a novel common analytical method and system by Reversed Phase High Performance Liquid Chromatography (RP-HPLC) for detection and quantification of melamine present in various Active Pharmaceutical Ingredients (APIs). Particularly, this invention relates to a unique, common analytical method and system for determination and quantification of melamine present in various categories of APIs as Cardiovascular, anti psychotic, stomach and esophageal, cardiovascular, anti-diabetic, antibiotic, anti-fungal, anti- gout, antimalarial, anti-Alzheimer and antiadrenergic categories.

Background of Invention:
Melamine, having following chemical formula, is chemically known as 1,3,5-Triazine-2,4,6-triamine. It is also known by other chemical names, such as, triaminotriazine, cyanuramide and cyanuric acid amide. It is a nitrogen-based compound that is characterized by its high nitrogen content. Melamine contains 66% nitrogen by molecular weight.

Melamine

Melamine is an industrial chemical used in the manufacturing of resins for surface laminates and adhesives in the production of wood-based panels. Melamine or its resins are also used in making melamine dinnerware, additives for textiles, and as flame retardant additives for foam mattresses.

The USFDA Guidance for Industry, “Pharmaceutical Components at Risk for Melamine Contamination” states and defines “components at risk for Melamine Contamination as “those ingredients or raw materials that rely on a test for nitrogen content for their identity or purity or strength, and that contain nitrogen in amounts greater than 2.5 percent w.r.t. molecular weight of the drug substances, such a component can be derived from source material that might be contaminated with melamine, or the component itself can, directly, be contaminated by melamine in an attempt to reduce its quality or strength or to substitute for it”. Contamination of any type in drugs can result in the loss of active ingredient strength and potency and can disrupt the processing of the finished product. Contaminants can also affect product or component stability. Any of these results from contamination can lead to reduced therapeutic effectiveness. Decreased drug effectiveness and possible long-term exposure risks raise serious concern over the safety of drugs contaminated with melamine and its analogs.

The test method used should be suitable for detecting melamine contamination in “at-risk components” down to at least 2.5 parts per million (ppm) to give a high degree of assurance that they are not contaminated.

As per the literature survey, Huaxue (2013), 41(2), 283-287, Rapid Communications in Mass Spectrometry (2016), 30(11), 1265-1272 reported determination of melamine content in milk/milk products by LC-MS/MS which is expensive and involves extensive sample preparation procedures.

However, some references also used High Performance Liquid Chromatography (HPLC) for determination of melamine content. Der Pharma Chemica, 2012, 4 (2):737-748 reported analysis of melamine in milk products using Symmetry C18 column (5 um, 250x 4.5 mm). Mobile phase is prepared by dissolving Buffer of citric Acid 2.10 gm and 2.16 gm sodium 1-octane sulfonate in 980 ml dionized water and two ranges of pH were tested at 4.5 and 3.0 and the volume brought to one liter after the pH adjustment by sodium hydroxide. Then 920 ml of the list solution was taken and mixed with 80 ml Acetonitrile. So the ratio is 92:8 volume to volume used for the isocratic separation mode for melamine.

Another journal, Guangdong Yaoxueyuan Xuebao, Volume20, Issue3, Pages236-237, 2004 reported detection of melamine in metformin hydrochloride using SCX column (250 mm×4.6 mm, 5 µm) as stationary phase, 1.7% ammonium dihydrogen phosphate (pH 3.0 adjusted with phosphoric acid) as mobile phase and at detection wavelength of 289 nm.

Yet another journal, Nanjing Gongye Daxue Xuebao, Ziran Kexueban, Volume37, Issue6, Pages131-134, 2015 reported detection of melamine in lansoprazole using SHISEIDO CAPCELL PAK 150 mm × 4.6 mm, 5 µm and the mobile phase was trifluoroacetic acid (TFA)-formic acid-water (0.05:0.3:100).

It is evident from the literature survey that for different API’s, different HPLC columns and different mobile phases are required to be used to determine the melamine content. This is major problem in pharmaceutical industry as pharma companies are involved in the manufacture of many active pharmaceutical ingredients (APIs) of various therapeutic categories. After checking melamine content in one API, the chemist has to change the column and mobile phase, flush the column and stabilize the system before checking melamine content in another API. This is a tedious process and consumes enormous amount of time leading to delay in dispatches of finished goods. The problem will be compounded with the increasing number of APIs manufactured by one company.

Therefore, there is a need to avoid such tedious process of changing columns and mobile phases frequently for detecting the presence of melamine content in various categories of API’s and to avoid consequent delays of dispatches of APIs. The present invention addresses the need and provides a common, single, analytical method and system for determination and quantification of melamine content in various categories of APIs.

Summary of Invention:
Accordingly, the present inventors have, surprisingly, developed a novel common analytical method and system by employing Reversed Phase High Performance Liquid Chromatography (RP-HPLC) to detect the presence of melamine in various Active Pharmaceutical Ingredients (APIs). This unique common method/system eliminates problems involved in practicing different methods for different APIs.

In an aspect, the invention provides a unique analytical system useful for determination and quantification of melamine present in various Active Pharmaceutical Ingredients (APIs), which system comprises a column of C18, (250mm × 4.6 mm, 5 µm) and mobile phase A and B wherein the mobile phase A is tetrabutyl ammonium hydroxide solution and the mobile phase B is Acetonitrile.

In another aspect, the invention provides an analytical method to detect and quantitate melamine content in a sample of Active Pharmaceutical Ingredients (APIs), which method comprises;
a) subjecting the sample to chromatography on a column of C18, (250mm × 4.6 mm, 5 µm);
b) eluting the sample with mobile phase A and B; and
c) determining the quantity of melamine in the sample;
wherein the mobile phase A is aqueous solution comprising tetrabutyl ammonium hydroxide and the mobile phase B is Acetonitrile.

In a preferred embodiment, the analytical method for determination and quantification of melamine present in various Active Pharmaceutical Ingredients (APIs) comprises a column of C18, (250mm × 4.6 mm, 5 µm) having more than 16% carbon loading and mobile phase A and B wherein the mobile phase A is tetrabutyl ammonium hydroxide solution and the mobile phase B is Acetonitrile.

In a more preferred embodiment, the analytical method for determination and quantification of melamine present in various Active Pharmaceutical Ingredients (APIs) comprises a column of C18, (250mm × 4.6 mm, 5 µm) having more than 16% carbon loading and mobile phase A and B wherein the mobile phase A is aqueous solution comprising tetrabutyl ammonium hydroxide buffer and the mobile phase B is Acetonitrile using gradient elution according to the following elution program, wherein the proportions of the mobile phases are all in volume percentages:

Time (min.) Flow (ml/min) Mobile Phase A (%) Mobile Phase B (%)
0.0 1.0 100.0 0.0
10.0 1.0 100.0 0.0
15.0 1.0 20.0 80.0
25.0 1.0 20.0 80.0
30.0 1.0 100.0 0.0
50.0 1.0 100.0 0.0

Brief description of tables and drawings:
Table-1 : Limit of detection and Limit of quantification values of Melamine
Table-2 : Accuracy details of Melamine
Figure-1: Limit of detection and Limit of quantification details
Figure-2: linerarity details of Melamine
Figure-3: Blank chromatogram
Figure-4: Melamine standard chromatogram
Figure-5: Atenolol test chromatogram
Figure-6: Atenolol test spiked at LOQ level chromatogram
Figure-7: Atenolol test spiked at 80% level chromatogram
Figure-8: Atenolol test spiked at 100% level chromatogram
Figure-9: Atenolol test spiked at 150% level chromatogram
Figure -10: Trimethoprim Test solution
Figure -11: Trimethoprim Test solution spike at LOQ level
Figure -12:Trimethoprim Test solution spike at 80% level
Figure -13:Trimethoprim Test solution spike at 100% level
Figure -14: Trimethoprim Test solution spike at 150% level
Figure -15: Midodrine Hydrochloride Test solution
Figure -16: Midodrine Hydrochloride Test solution spike at LOQ level
Figure -17: Midodrine Hydrochloride Test solution spike at 80% level
Figure -18 Midodrine Hydrochloride Test solution spike at 100% level
Figure -19: Midodrine Hydrochloride Test solution spike at 150% level
Figure -20: Chlorthalidone Test solution
Figure -21: Chlorthalidone Test solution spike at LOQ level
Figure -22: Chlorthalidone Test solution spike at 80% level
Figure-23: Chlorthalidone Test solution spike at 100% level
Figure -24: Chlorthalidone Test solution spike at 150% level
Figure-25: Quetapine Fumarate Test solution
Figure -26: Quetapine Fumarate Test solution spike at LOQ level
Figure -27: Quetapine Fumarate Test solution spike at 80% level
Figure -28: Quetapine Fumarate Test solution spike at 100% level
Figure -29: Quetapine Fumarate Test solution spike at 150% level

Description of Invention:
The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

Unless specified otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, to which this invention belongs. To describe the invention, certain terms are defined herein specifically as follows.

Unless stated to the contrary, any of the words, “including”, “includes”, “comprising”, and comprises” mean “including without limitation” and shall not be construed to limit any general statement that it follows to the specific or similar items.

Accordingly, the present inventors have, surprisingly, developed a novel common analytical method and system by employing Reversed Phase High Performance Liquid Chromatography (RP-HPLC) for determination and quantification of melamine present in various Active Pharmaceutical Ingredients (APIs).

According to one embodiment, the invention provides a unique analytical system for determination and quantification of melamine present in various Active Pharmaceutical Ingredients (APIs) which comprises a column of C18, (250mm × 4.6 mm, 5 µm) and mobile phase A and B wherein the mobile phase A comprises aqueous solution of tetrabutyl ammonium hydroxide buffer and the mobile phase B is Acetonitrile.

In another embodiment, the invention provides an analytical method to detect and quantitating melamine content in a sample of Active Pharmaceutical Ingredients (APIs), which method comprises;
a) subjecting the sample to chromatography on a column of C18, (250mm × 4.6 mm, 5 µm);
b) eluting the sample with mobile phase A and B; and
c) determining the quantity of melamine in the sample;
wherein the mobile phase A is aqueous solution comprising tetrabutyl ammonium hydroxide and the mobile phase B is Acetonitrile.

Usually the analysis is carried out on Waters HPLC pump - alliance (2695), auto sampler- alliance (2695); equipped with PDA detector with empower software. A C18 reversed phase column having length of 250 mm, particle size of 5 µm and diameters of 4.6 mm (250mm × 4.6 mm, 5 µm) of specifications are used in the analysis.

In a preferred embodiment, the analytical method and system for determination and quantification of melamine present in various Active Pharmaceutical Ingredients (APIs) comprises use of a column of C18, (250mm × 4.6 mm, 5 µm) having more than 16% carbon loading for analysis. Typically Unisphere Extend C18 (250mm × 4.6 mm, 5 µm) column made by Agela Technologies is used.
A combination of two mobile phases, mobile phase A and B, are used wherein the mobile phase A is tetrabutyl ammonium hydroxide solution and the mobile phase B is Acetonitrile. Typically tetrabutyl ammonium hydroxide is commercially available as 0.1N solutions in alcohols such as isopropanol and this iso-propanol solution is mixed with water to obtain 0.08 to 0.12 % V/V concentration of tetrabutyl ammonium hydroxide aqueous solution. Alternately, the tetrabutyl ammonium hydroxide may be dissolved in water directly to make tetrabutyl ammonium hydroxide aqueous solution. Preferred concentration of the mobile phase A, tetrabutyl ammonium hydroxide aqueous solution, is 0.1% V/V.

In a more preferred embodiment, the analytical method and system for determination and quantification of melamine present in various Active Pharmaceutical Ingredients (APIs) comprises a column of C18, (250mm × 4.6 mm, 5 µm) having more than 16% carbon loading and mobile phase A and B wherein the mobile phase A is aqueous solution comprising tetrabutyl ammonium hydroxide buffer and the mobile phase B is Acetonitrile using gradient elution according to the following elution program, wherein the proportions of the mobile phases are all volume percentages:

Time (min.) Flow (ml/min) Mobile Phase A (%) Mobile Phase B (%)
0.0 1.0 100.0 0.0
10.0 1.0 100.0 0.0
15.0 1.0 20.0 80.0
25.0 1.0 20.0 80.0
30.0 1.0 100.0 0.0
50.0 1.0 100.0 0.0

This analytical method is suitable for determination and quantification of melamine present in various categories of APIs. The various categories of APIs include but not limited to cardiovascular, anti psychotic, antiulcerative, antiemetic, anti-diabetic, antibiotic, anti fungal, anti gout, antimalarial, anti-Alzheimer and antiadrenergic categories. The APIs include but not limited to Midodrine Hydrochloride, Atenolol, Chlorthalidone, Amlodipine Besylate, Valsartan and Clopidogrel Bisulphate from cardiovascular category, Quetiapine Fumarate, Olanzapine, Sumatriptan Succinate, Venlafaxine Hydrochloride, Eszopiclone, Zopiclone and Lamotrigine from anti psychotic category, Famotidine from antiulcerative category, Metoclopramide Hydrochloride from antiemetic category, Glimepiride and Gliclazide from Anti-diabetic category, Trimethoprim from antibiotic category, Fluconazole from Anti-fungal category, Allopurinol from antigout category, Hydroxy Chloroquine sulphate from Antimalarial category, Donepezil Hydrochloride Anti-Alzheimer category and Silodocin from Antiadrenergic category.

According to the invention, Melamine content can be determined in multiple API’s within a short span of time. The chromatographic conditions i.e. Column, mobile phase, standard preparation and sample preparation steps are identical for all the mentioned APIs belonging to various categories.
The steps involved in analysis are mentioned below:
1. During the analysis, the diluent i.e. Mobile phase A is injected and no interference should be obtained at the Retention Time of Melamine peak.
2. Melamine Standard solution (0.10 ppm) is injected in six replicates and it’s average area is to be considered for quantification of Melamine in various APIs.
3. Test solution of various categories of APIs are prepared in identical manner in diluent i.e. Mobile phase A, shaken and sonicated for 2.0 minutes and then filtered through 0.45µ nylon syringe filter. Only the Melamine is dissolved in the Mobile Phase A & not the water insoluble API.
4. Test solution of individual API’s belonging to different categories are injected to determine the melamine content in individual API’s. Bracketing standard of Melamine can be injected in-between for checking consistency and to ensure integrity over a course of time.
From the above, in single day multiple API samples can be analyzed for determining the presence or absence of Melamine in continuous process with precise and accurate results. And, there is no time consumed in changing the column and stabilizing the HPLC system since this single system and method has the capability for detecting and quantifying Melamine in different API’s.
Details of steps involved in the analysis method are explained below.

The following examples along with detailed analytical procedures and system are presented below to further explain the invention which are purely illustrative and are not intended to limit the scope of the claimed invention.

Example 1
Preferred chromatographic conditions:
Analytical column Unisphere Extend C18 (250mm x 4.6 mm, 5µm)
Detection wavelength 220 nm
Flow rate 1 mL/min
Column oven temperature 30°C
Injection volume 50µl
Run time 50 min
Diluent Mobile phase A & B
Mobile phase A:-0.1%v/v Tetra butyl Ammonium Hydroxide 0.1N solution in Isopropyl Alcohol in water &
B:-Acetonitrile

Gradient program:
Time (min.) Flow (ml/min) Mobile Phase A (%) Mobile Phase B (%)
0.0 1.0 100.0 0.0
10.0 1.0 100.0 0.0
15.0 1.0 20.0 80.0
25.0 1.0 20.0 80.0
30.0 1.0 100.0 0.0
50.0 1.0 100.0 0.0

Melamine Retention Time is observed at about 4.9 min.

Example 2
Preparation of Standard stock solution of Melamine:
Standard stock solution is prepared by weighing 10 mg of melamine standard in 100 mL of volumetric flask, added about 30 mL of diluent, sonicated to dissolve the melamine and diluted up to the mark with diluent. Transferred 1.0 mL of this solution into 100 mL volumetric flask and diluted up to the mark with diluent. (Concentration of Melamine is 1.0 ppm)

Preparation of Melamine Standard solution:
Transferred 1.0 mL of Standard stock solution into 10 mL of volumetric flask and diluted up to the mark with diluent. (Concentration of Melamine is 0.1 ppm)
Melamine Standard solution is used as System suitability solution.

Example 3
Test preparation:
Test solution is prepared by weighing 400 mg of the active pharmaceutical substance in 10 mL of volumetric flask and diluted up to the mark with diluent. Shaken and sonicated for 2.0 minutes and then filtered through 0.45µ nylon syringe filter.
Validation of Proposed Method:
The method is validated as per the ICH guidelines.

Example 4
Limit of detection (LOD) and Limit of quantitation (LOQ):
The limit of detection (LOD) and quantification (LOQ) is determined by injecting a series of diluted solution of Melamine having concentration close to theoretical limit of detection and quantification. The limit of detection of Melamine is established based on standard deviation of response and the slope (as per ICH guideline).

The detection limit (DL) of Melamine is calculated from the calibration curve using the formula i.e. DL= [3.3 x Standard error/Slope].

The limit of quantitation (QL) of Melamine is calculated from the calibration curve using the formula i.e. QL= [l0 x Standard error /Slope].

The LOQ level of melamine is injected six times and %RSD (relative standard deviation) of area of replicate injections is calculated and found to be 0.36%, which is well within the limit of RSD NMT 10.00%.

LOD and LOQ for Melamine obtained are mentioned in tabulated form as shown below in table 1. Limit of detection (LOD) and Limit of quantification (LOQ) details are also shown in figure 1.

Table 1: Limit of detection (LOD) and Limit of quantification (LOQ) values of Melamine
Name of component Limit of Detection Limit of Quantification
Melamine Conc as such (in ppm) Conc w.r.t Test solution
(in ppm) Conc as such (in ppm) Conc w.r.t Test solution
(in ppm)
0.0011 0.0275 0.0032 0.0800

Linearity:
Linearity is determined by serial dilution of Melamine to 6 different concentrations in the range of LOQ to 150% of the impurity limit. The response is recorded as peak areas and plotted against the concentration. Correlation coefficient, slope and intercept were calculated. For Melamine, the correlation coefficient is obtained as 1.0000 showing that response is linear. Linearity details of Melamine are shown in figure 2.
Accuracy:
Accuracy is performed at LOQ level, 80%, 100% and 150% of the melamine impurity limit.
From the data obtained, % recovery of melamine is calculated against added amount.
Recovery of Melamine is performed in all the categories of API, however the results of API belonging to three different categories are mentioned below in table 2.

Table 2: Accuracy details of Melamine
Category of API Name of API Mean % recovery at LOQ level Range of %Recovery obtained for 80%, 100% and 150% spiked conc
For Cardiovascular treatment Atenolol 105.77% 105.46% to 108.49%
Antibiotic Trimethoprim 95.79% 100.42% to 101.77%
Anti psychotic Venlafaxine Hydrochloride 89.65% 93.24% to 93.70%

Further, chromatograms of test solutions of various active pharmaceutical ingredients such as atenolol, Trimethoprim, Midodrine, Chlorthalidone, Quetapine Fumarate and the test solution spiked with melamine concentration at LOQ level, 80%, 100% and 150% are shown in figures 5 to 29.