FIELD OF THE INVENTION
The present invention relates to a new, robust, economical and industrially viable Reverse Phase-High performance liquid chromatography (RP-HPLC) method for the determination and quantification of genotoxic nitrosamine impurities in drug substance or drug products or their combination products up to nano-gram level.
BACKGROUND OF THE INVENTION
Nitrosamines are chemical compounds classified as probable human carcinogens on the basis of animal studies. They can cause DNA mutations which are potentially leading to cancer.
Nitrosamines, or more correctly N-nitrosoamines, refer to any molecule containing the nitroso functional group. These molecules are of concern because nitrosamine impurities are probable human carcinogens. Although they are also present in some foods and drinking water supplies but their presence in medicines is nonetheless considered unacceptable. These compounds are commonly found in grains, cured meat, beer, tobacco and drinking water as well as being intermediates in organic synthesis. Several medications have been recalled due to the presence of nitrosamine compounds as impurities in the final drug products.
FDA has identified seven nitrosamine impurities that can be present in drug products: N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitroso-N-methyl-4-aminobutanoic acid (NMBA), N-nitrosoisopropylethyl amine (NIPEA), N-nitrosodiisopropylamine (NDIPA), Nnitrosodibutylamine (NDBA), and N-nitrosomethylphenylamine (NMPA).
In year 2018, U.S. FDA issued warning letters to the drug product of Valsartan, as the FDA identified nitrosamine impurities N-Nitrosodimethylamine (NDMA) and N-Nitrosodiethylamine (NDEA) in active pharmaceutical ingredient (API) and finished drugs. Valsartan is an Angiotensin II Receptor Blocker (ARB) and belongs to a family of analogue compounds commonly referred to as the Saltans. The recalled products contain the levels of NDMA or NDEA exceeding the interim Acceptable Intake (AI) limits.
More recently, nitrosamine impurities have been reported in pioglitazone and ranitidine containing products.

nitrosodimethylamine In year 2020, FDA issue warning letters Ranitidine, a histamine-2 blocker. FDA has found N- (NDMA) levels in some ranitidine products increase with time and temperature posing a risk to consumers. FDA recommends the following Acceptable Intake (AI) limits for the nitrosamine impurities NDMA, NDEA, NMBA, NMPA, NIPEA, and NDIPA (Table 1).

S.No. Nitrosamine Structure with their names Nitrosamine AI
Impurities Abbreviations Limit (ng/day)
N-NL 96
1. NDMA N-Nitrosodimethylamine (NDMA)
2. NDEA N-NL
o* ^-
N-Nitrosodiethylamine (NDEA) 26.5
3. NMBA 0
AoH 96
N-Nitroso-N-methyl-4-aminobutyricacid (NMBA)
(^\
4. NMPA u ^N'% 26.5
N-Nitrosomethylphenylamine (NMPA)
5. NIPEA N-NL
o* ^-
N-Nitrosodiisopropylethylamine (NIPEA) 26.5
6. NDIPA 26.5

N-Nitrosodiisopropylamine (NDIPA)

7. NDBA N-Nitrosodibutylamine (NDBA) Not mentioned
Table 1: Chemical Structures of Seven Potential Nitrosamine Impurities and their AI Limits in Drug Products
The formation of nitrosamines is generally only possible when secondary or tertiary amines react with nitrous acid. Nitrous acid itself is unstable but can be formed in situ from nitrites (NO2) under acid conditions.
In the case of the Sartan compounds, most contain a tetrazole ring and formation of this tetrazole ring employs the use of sodium nitrite. Coincidently the solvents employed either were amines, or contained traces of amines, and this likely afforded the observed NDMA and NDEA. The origins of NDMA content in batches of ranitidine currently remains unclear.
However, during on-going investigations it was also concluded that the possibility for nitrosamine impurity content was broader than simply the concurrent presence of nitrites and amines in the synthesis of the active pharmaceutical ingredient (API).
NDMA and NDEA are classified as a probable human carcinogen based on the results from laboratory animal tests and are listed under WHO/IARC group 2A and EPA group B2. NDMA and NDEA contamination was thought to be caused by the changes in the production process in case of Valsartan API.
Gas chromatography-mass spectrometry (GC-MS) is the most frequently employed technique for nitosamine impurities analyses. The FDA Office of Testing and Research have developed a combined GC/MS headspace method for the simultaneous evaluation of four nitrosamine impurities in angiotensin II receptor blockers (ARB) drug substance and drug product. These impurities are; N-nitrosodimethyl amine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodiisopropylamine (NDIPA), and N-nitrosoethylisopropylamine (NEIPA). The method was developed and validated on valsartan drug substance and drug product.

In addition, several methods using liquid chromatography-mass spectrometry (LC-MS) have been reported in scientific literature. The FDA has observed that the method for testing angiotensin II receptor blockers (ARBs) for nitrosamine impurities is not suitable for testing Ranitidine because heating the sample generates NDMA.
A LC-HRMS method was subsequently developed by the FDA to measure the levels of NDMA in ranitidine drug substance and drug product following ICH Q2(R1), with LOD lOng/g, lower LOQ 33ng/g and upper LOQ 3333ng/g.
The use of GC/MS or LC-HRMS method suggested by FDA is on trial as it does not give a clear picture about detection of all seven nitrosamine impurities in other drug substance and drug products. Only few studies have reported NDMA/NDEA analysis using conventional high-performance liquid chromatography (HPLC), especially in drugs. HPLC is the most popular technique for quality control of APIs and products in routine analysis.
It is preferable if NDMA and NDEA impurities are simultaneously detected within the drug substance as well as drug product by a single HPLC analysis.
The low levels at which the nitrosamine impurities occur creates challenges for testing. Hence, there is a long felt need to develop an economically viable method using HPLC. To assist in the testing of samples the inventors of the present invention have developed a novel and robust quantification method of analysis for major nitrosamine impurities i.e. NDMA and NDEA in various drug substances (API) as well as in drug products by using High-performance liquid chromatography (HPLC).
SUMMARY OF THE INVENTION
The invention relates to a robust, industrially viable and cost effective high pressure liquid chromatography (HPLC) method for determining the genotoxic nitrosamine impurities in drug substance or drug product or their combination products comprising the steps of:
a) preparing sample solution comprising drug substance or drug product or their combination products in diluent
b) optionally vortex mix and centrifuge or sonicate
c) filter the solution of step b)
d) performing HPLC using chromatographic conditions:

Input Parameters (range)
Mobile Phase (A:B)% (v/v) 80-95:5-20
Elution mode Isocratic
Column Specification Cig (250X4.6)mm,5um
Wavelength (nm) 225-229
Column Temperature (°C) 35-40
Flow Rate (ml/min) 0.5-1.0
Run Time (min.) 20-65
Linearity (ng/ml) 5-1.1
LOD (ng/ml) 0.5
LOQ (ng/ml) 1.1
Recovery (%) 80-120
e) performing step a) to d) for standards of nitrosamines either
individual or in groups of nitrosamines. f) determining the quantitative values of nitrosamines up to a precision of 1 nanogram level. The invention also relates to a robust, industrially viable and cost effective high pressure liquid chromatography (HPLC) method, where the genotoxic nitrosamine impurities consist of N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosoethylisopropylamine (NEIPA), N-nitrosodiisopropylamine (NDIPA), N-nitrosodibutylamine (NDBA), N-nitrosomethyl-4-amino-butyric acid (NMBA), in drug substances and drug products or their combination products.
BRIEF DESCRIPTION OF THE DRAWINGS:
FIG.l: is an illustration of a High performance Liquid Chromatography ("HPLC") of Omeprazole - Drug Product obtained according to the present invention.
FIG.2: is an illustration of a High performance Liquid Chromatography ("HPLC") of Omeprazole Placebo.
FIG.3: is an illustration of a High performance Liquid Chromatography ("HPLC") of Ibuprofen NDMA - Drug Substance according to the present invention.
FIG.4: is an illustration of a High performance Liquid Chromatography ("HPLC") of Ibuprofen NDEA - Drug Substance according to the present invention.

FIG.5: is an illustration of a High performance Liquid
Chromatography ("HPLC") of Paracetamol NDMA - Drug Substance
according to the present invention.
FIG.6: is an illustration of a High performance Liquid
Chromatography ("HPLC") of Paracetamol NDEA - Drug Substance
according to the present invention.
FIG.7: is an illustration of a High performance Liquid Chromatography ("HPLC") of all six Nitrosamine impurities according to the present invention.
DETAILED DESCRIPTION:
Embodiments according to the present invention relates to a robust, industrially viable and cost effective high pressure liquid chromatography (HPLC) method for determining the genotoxic nitrosamine impurities in drug substance or drug product or their combination products comprising the steps of:
a) preparing sample solution comprising drug substance or drug product or their combination products in diluent
b) optionally vortex mix and centrifuge or sonicate
c) filter the solution of step b)
d) performing HPLC using chromatographic conditions:

Input Parameters (range)
Mobile Phase (A:B)% (v/v) 80-95:5-20
Elution mode Isocratic
Column Specification Cig (250X4.6)mm,5um
Wavelength (nm) 225-229
Column Temperature (°C) 35-40
Flow Rate (ml/min) 0.5-1.0
Run Time (min.) 20-65
Linearity (ng/ml) 5-1.1
LOD (ng/ml) 0.5
LOQ (ng/ml) 1.1
Recovery (%) 80-120

e) performing step a) to d) for standards of nitrosamines either individual or in groups of nitrosamines.
f) determining the quantitative values of nitrosamines up to a precision of 1 nanogram level.
The present invention is particularly suitable for determining and quantifying the presence of one or more of genotoxic nitrosamine impurities selected from N-nitrosodimethyl amine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosoethylisopropylamine (NEIPA), N-nitrosodiisopropylamine (NDIPA), N-nitrosodibutylamine (NDBA), N-nitrosomethyl-4-amino-butyric acid (NMBA), in drug substances and drug products or their combination products..
In step a) of preparing the sample solution comprises the steps of, weighing the sample comprising drug substance or drug product or their combination products and adding diluent to the weighed sample followed by mixing.
The diluent medium used in step ii) of sample preparation is selected from perchloric acid, MilliQ water and methanol or in combination thereof. The role of diluent is critical as the sample must be soluble in the diluent and do not react with any of the diluent components. Determination of the solution stability in the diluent is also important during early method development.
Inventors of the present invention have found perchloric acid, MilliQ water and methanol or in combination thereof are most suitable diluent for the sample which are analyzed on the developed reverse phase HPLC method as the samples are completely soluble in it and the resulting solution are clear in nature.
Inventors also found the diluent selected are compatible with the mobile phase to obtain the good peak shape.
In another embodiment of the present invention, the sample solution prepared is optionally vortex mix for performed for 10-20 minutes more preferably for lOminutes followed by centrifuge or sonicate for 30-60minutes at 4000rpm.
In the prior art the methods for extraction involves solid-phase extraction, solid-phase micro-extraction (SPME), autoclave extraction and supercritical fluid extraction (SFE).
In the present invention, inventors have found initial vortex mixing and later centrifugation is sufficient for extraction of targeted compound for reverse phase HPLC analysis.

The vortex mixing for 10 minutes and centrifugation process for 30 minutes results in better extraction because vortex mixing initiate the movement of particles and in centrifugation the particles of the sample are moved at a very high speed while the other extraction are time taking, tough to perform in daily analytical practices.
In another embodiment of the present invention, the sample is filtered after centrifugation from a 0.2um nylon/mdi/PVDF/PTFE/Glass filter.
Filtration of sample is required for the removal of solid particulate as the solid particulate may block the HPLC column and damage the HPLC system while sample run. Inventors found 0.2um nylon filter suitable for the sample filtration, due to its hydrophilic nature, flexibility, tear-resistant ability, affordability, can be used small volume filtration and not required any major or heavy equipment for filtration.
In prior known literature the methods involves for the detection of nitrosamine impurities involves calorimetry, spectrophotometry, Micellar electro-kinetic capillary chromatography, Gas chromatography with flame ionization detection, nitrogen phosphorous detection, thermal energy detection, nitrogen chemilumines detection and mass spectrometry detection, High-performance liquid chromatography with thermal energy analyzer, mass spectrometry and fluorescence detection, High-performance liquid chromatography (HPLC)- chemiluminescence method, and chromatography-mass spectrometry.
The known methods are limited to the analysis of some specific nitrosamine impurities in selected compounds. No method is sufficient to detect and quantify the six nitrosamine in different drugs substance and drug products or their combination thereof. The prior reported method is mostly performed on GC-MS which is high in cost and lesser reliable in terms of peak separation as compared to the reverse phase HPLC method of the present invention.
The current invention is also advantageous as the method is selective, sensitive, linear, precise, accurate and robust for the analysis of genotoxic nitrosamine impurities in drug substance or drug product or their combination products. In addition, the current invention is highly sensitive and allows detection and quantification of genotoxic nitrosamine impurities in drug substance or drug product or their combination products at levels much lower than acceptance limits specified by health authorities and in ICH Guidelines.
In another embodiment the sample prepared is run on the reverse phase HPLC with developed method.

The HPLC which is used by the inventors is of brand Schimadzu.
The chromatographic conditions developed for the determination of genotoxic nitrosamine impurities in drug substance or drug product or their combination products are mentioned below in the table.

Input Parameters (range)
Mobile Phase (A:B)% (v/v) 80-95:5-20
Elution mode Isocratic
Column Specification Cig (250X4.6)mm,5um
Wavelength (nm) 225-229
Column Temperature (°C) 35-40
Flow Rate (ml/min) 0.5-1.0
Run Time (min.) 20-65
Linearity (ng/ml) 5-1.1
LOD (ng/ml) 0.5
LOQ (ng/ml) 1.1
Recovery (%) 80-120
In the present invention the HPLC method validated as per ICH Q2A Guidelines covering the parameters Specificity, Linearity and Range, Precision (Repeatability, Reproducibility and Intermediate Precision), Accuracy, Limit of Detection (LOD), Limit of Quantitation (LOQ), Robustness and System Suitability.
In the working of the invention the inventors of the present invention have found that stationary phases particularly preferred a Chromcore (250 mm x 4.6 mm), 5 urn column.
The method of the current invention preferably comprises a isocratic mode as Isocratic elution exhibits greater simplicity, lower cost, simpler instrumentation, and no need of column re-equilibration between consecutive injections than the gradient mode.
The mobile phase used is designated as Mobile Phase A and Mobile Phase B. The mobile phase A is preferably water and 0.1 % of an organic acid. In the present invention the organic acid used in mobile phase A is trifluoroacetic acid. The Mobile Phase B is selected from acetonitrile or methanol.
In the prior art the method of HPLC performed for nitrosamine impurities use many combination of mobile phase A as methanol and mobile phase B as water. However, inventors have performed and found that the know methods are useful for

the higher values of detection and can be used to quantify at a high concentration. The recovery in the prior art is also low as per the ICH guidelines.
Typically, the method of the current invention is carried out at a column temperature between approximately 35 to 40°C at a wavelength of 225-229nm.
Inventors analyses the elution of the peak should be sharp and does not overlap or merge with the any other peaks. At a wavelength of 228nm inventors precisely distinguish between the nitrosamine impurities and the other peaks of drug substance or drug product or their combination products.
Flow rate in the present invention is set about 0.5-1.0ml/min and the samples are run 20-65 minutes.
The method developed and validated by the inventors is having the Limit of detection of 0.5 ng/ml and limit of quantification can measure up to l.lng/ml i.e. the developed method is so precise for the detection of genotoxic nitrosamine impurities up to 0.5ng/ml level and can be used to quantify in l.lng/ml in the respective drug substance or drug product or their combination products.
The inventors of the present invention have found the recovery level of the said RP-HPLC method is in the range of 80-120%.
In a particular embodiment according to the present invention the recovery level was found to be in the range of 85-115%.
In another embodiment, the standards of nitrosamine impurities are also tested over the developed RP-HPLC method. The results show a clear, precise separation of all six genotoxic nitrosamine impurities.
The present disclosure is further described in light of the following experiments which are set forth for illustration purpose only and not to be construed for limiting the scope of the disclosure. The method developed can be extrapolated to industrial scale. EXAMPLES
The RP-HPLC method for the analysis of genotoxic nitrosamine impurities in drug substance or drug product or their combination products were based on Cig reversed phase HPLC with UV-Visible detection. Example-1 Omeprazole Drug Product
Buffer: Accurately transfer 1.0 ml Trifluoroacetic acid in 1000 ml MilliQ water, mix and sonicate. Filtered through 0.45 um membrane filter and degas. Preparation of Mobile phase-A: Buffer (100) % Preparation of mobile phase B: Acetonitrile (100) %

Diluent preparation: Dilute 10 ml of Perchloric acid in 1000 ml of MilliQ water and
mix.
Chromatographic conditions:

Column : ChromCore PFP (250 x 4.6)mm, 5 um or equivalent
Flow : 0.5 mL/minutes
Wavelength : 228nm
Injection Volume : 200 uL
Column oven Temperature :40°C
Sample cooler Temperature : 5°C
Run Time : 65 min.
Reagents:
1. Methanol, HPLC grade
2. Trifluoroacetic acid, AR grade
3. MilliQ water
4. Perchloric acid, AR grade
5. Acetonitrile, HPLC grade
6. Solvent/Diluent: Mix 10ml of Perchloric acid in 1000ml of MilliQ water
7. N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA) reference standard
Preparation of Standard Solution:
a) Stock Solution 1: Weigh and transfer about 20mg of NDMA & NDEA working standards into a 200ml volumetric flask, add about 60ml of diluent and sonicate for 5minutes to dissolve. Makeup the volume with diluent and mix.
b) Stock Solution 2: To the 1ml of stock solution 1 add 200ml of diluent and mix.
c) Standard Solution: To the stock solution 2 add 50ml of diluent and mix.
Preparation of Sample Solution: Weigh and transfer equivalent to about 30 mg of Omeprazole into 2ml of diluent. The mixture is vortex mix for about lOminutes and centrifuge for about 30 minutes at 4000rpm. Resulting solution filter through 0.2um nylon/mdi filter. System Suitability:

1. Replicate injections of quantitation limit solutions must be
repeatable;
6 injections, RSD= 10%
Procedure: After system suitability criteria are met, proceed with the
standard and sample injections.
Calculations (For NDMA):
Calculate the percentage of NDMA impurity in Omeprazole by using the
formula:
AT WS P Average fill Weight
Impurity (%w/w) = X X X XI00
AS WT 100 Label Claim
Where,
AT: Peak area of known impurity in chromatogram obtained from sample solution.
AS: Average peak area of six replicate injections of NDMA in chromatogram
obtained from standard solution.
WS: Concentration of NDMA WS/RS in standard solution (mg/ml).
WT: Concentration of Omeprazole in sample solution (mg/ml).
P: Potency of NDMA WS/RS.
Calculation (For NDEA):
Calculate the percentage of NDEA impurity in Omeprazole by using the formula:
AT WS P Average fill Weight
Impurity (%w/w) = X X X XI00
AS WT 100 Label Claim
Where, AT: Peak area of known impurity in chromatogram obtained from sample solution.
AS: Average peak area of six replicate injections of NDEA in chromatogram
obtained from standard solution.
WS: Concentration of NDEA WS/RS in standard solution (mg/ml).
WT: Concentration of Omeprazole in sample solution (mg/ml).
P: Potency of NDEA WS/RS.
TESTING FOR NDMA

Drug Product Maximum Daily Dose (mg/day) Acceptable Intake NDMA (ng/day) Limits of NDMA (ppm)
Omeprazole Capsule 360 96 0.267

TESTING FOR NDEA

Drug Product Maximum Daily Dose (mg/day) Acceptable Intake NDEA (ng/day) Limits of NDEA (ppm)
Omeprazole Capsule 360 26.5 0.074
Example-2 Paracetamol Drug Substance Method-A : N-nitrosodimethylamine (NDMA)
Preparation of Mobile phase A: Water with 0.1% Tri fluoro acetic acid (TFA) by volume Preparation of mobile phase B: Acetonitrile. Diluent preparation: Use MilliQ water as diluent.

Column : ChromCore PFP (250 x 4.6)mm, 5 um or equivalent
Flow : 1.0 mL/minutes
Wavelength : 228nm
Injection Volume : 200 uL
Column oven Temperature :40°C
Sample cooler Temperature : 5°C
Run Time : 55 min.
Elution pattern (Binary/premix) Mobile phase A: Mobile phase B (93:07)% v/v.
Reagents:
1. Methanol, HPLC grade
2. Trifluoroacetic acid, AR grade
3. MilliQ water
4. Perchloric acid, AR grade
5. Acetonitrile, FtPLC grade
6. Solvent/Diluent: Mix 10ml of Perchloric acid in 1000ml of MilliQ water
7. N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA) reference standard
Preparation of Standard Solution:
d) Stock Solution 1: Weigh and transfer about 20 mg of NDMA working standard into a 200 ml volumetric flask, add about 60 ml of diluent and sonicate for 5 minutes to dissolve. Makeup the volume with diluent and mix.

e) Stock Solution 2: To the 1ml of stock solution 1 add 200ml of diluent and mix.
f) Standard Solution: To the stock solution 2 add 50ml of diluent and mix.
Preparation of Sample Solution: Weigh and transfer about 200 mg of drug substances into 2 ml of diluent. The mixture was vortex mixed for about 10 minutes and centrifuged for about 30 minutes at 4000 RPM. Resulting solution filtered through 0.2 um nylon/mdi filter.
Aliquots of the solution were transferred to HPLC vials for analysis by HPLC System Suitability:
1. Replicate injections of quantitation limit solutions must be
repeatable;
6 injections, RSD= 10% Procedure: Create sequence as follow and inject the solution according to the sequence.

S.No. Sample name No. of Injection
1 Blank (Diluent) 1
2 Standard Solution 6
3 Test Solution 1
4 Standard SolutionBkt 1
Relative retention time for impurity are as follows:

S.No. Name of the Standard/Impurity RRT(minute)
1 NDMA 0.65
2 Paracetamol 1.00
The retention time of Paracetamol is about 8.6 minutes and NDMA about 5.1 minutes. Calculation:
Calculate the percentage of known impurity in Paracetamol by using the formula:
AT WS P
Impurity (%w/w) = X X XI00
AS WT 100

Where,
AT: Peak area of known impurity in chromatogram obtained from sample solution. AS: Average peak area of six replicate injections of NDMA in chromatogram
obtained from sample solution. WS: Concentration of NDMA WS/RS in standard solution (mg/ml). WT: Concentration of Paracetamol in sample solution (mg/ml). P: Potency of NDMA WS/RS.
Method-B : N-nitrosodiethylamine (NDEA)
Preparation of Mobile Phase A: Water with 0.1% Tri Fluoro Acetic Acid (TFA) by volume
Preparation of Mobile Phase B: Acetonitrile.
Diluent preparation: Use MilliQ as diluent.
Chromatographic conditions:
Column oven Temperature : 35°C
Sample cooler Temperature : 5°C
Run Time : 45 min.
Elution pattern : Mobile phase A: Mobile phase B (80:20) %v/v.
(Binary/premix)
Preparation of Standard solution (O.Olppm): Accurately weight and transfer about 20 mg of NDEA working standard into a 200 ml volumetric flask, add about 60 ml of diluent and sonicate for 5 minutes to dissolve. Makeup the volume with diluent and mix (Stock solution-1). Further, dilute 1ml of stock solution-1 to 200ml with diluent and mix (Stock solution-2). Further, dilute 1.0 ml of stock solution-2 to 50 ml with diluent and mix (Standard solution).
Preparation of sample solution: Accurately weight and transfer about 400 mg of drug
substances into 2 ml of diluent. The mixture was vortex mixed for about 10 minutes and
centrifuged for about 30 minutes at 4000 RPM. Resulting solution filtered through 0.2 um
nylon/mdi filter.
Aliquots of the solution were transferred to FtPLC vials for analysis by FtPLC.
Procedure: Create sequence as follow and inject the solution according to the sequence.

S.No. Sample name No. of Injection
1 Blank (Diluent) 1
2 Standard Solution 6
3 Test Solution 1
4 Standard SolutionBkt 1

Relative retention time for impurities are as follows:

S.No. Name of the Standard/Impurity RRT(minute)
1 NDEA 1.62
2 Paracetamol 1.00
The retention time of Paracetamol is about 4.8 minutes and NDEA about 7.6 minutes.
System Suitability:
• % RSD of six replicate injections of standard solution should not be more than 10.0%.
Calculation:
Calculate the percentage of known impurity in Paracetamol by using the formula:
AT WS P
Impurity (%w/w) = X X X 100
AS WT 100 Where,
AT: Peak area of known impurity in chromatogram obtained from sample solution.
AS: Average peak area of six replicate injections of NDEA in chromatogram
obtained from sample solution.
WS: Concentration of NDEA WS/RS in standard solution (mg/ml).
WT: Concentration of Paracetamol in sample solution (mg/ml).
P: Potency of NDEA WS/RS.
TESTING FOR NDMA

Drug Substance Maximum Daily Dose (mg/day) Acceptable Intake NDMA (ng/day) Limits of NDMA (ppm)
Paracetamol 4000 96 0.024
TESTING FOR NDEA

Drug Substance Maximum Daily Dose (mg/day) Acceptable Intake NDEA (ng/day) Limits of NDEA (ppm)
Paracetamol 4000 26.5 0.007

Example 3 Ibuprofen Drug Substance
Buffer: Accurately transfer 1.0 ml Trifluoro acetic acid in 1000 ml MilliQ water, mix and sonicate. Filtered through 0.45 um filter paper and degas. Preparation of Mobile phase-A: Buffer (100) % Preparation of mobile phase B: Acetonitrile (100) % Diluent preparation: MilliQ water (100) % Chromatographic conditions:

Column : ChromCore PFP (250 x 4.6)mm, 5 um or equivalent
Flow : 1.0 mL/minutes
Wavelength : 228nm
Injection Volume : 200 uL
Column oven Temperature :40°C
Sample cooler Temperature :5°C
Run Time : 55 min.
Elution pattern (Binary/premix) Mobile phase A: Mobile phase B (92:08) %v/v.
Preparation of Standard Solution:
1. Stock Solution 1: Weight and transfer about 20 mg each of NDMA working standards into a 200 ml volumetric flask, add about 60 ml of diluent and sonicate for 5 minutes to dissolve. Makeup the volume with diluent and mix
2. Stock Solution 2: dilute 1ml of stock solution-1 to 200ml with diluent and mix
3. Standard Solution: dilute 1.0 ml of stock solution-2 to 50 ml with diluent and mix
Preparation of sample solution (For NDMA): Accurately weigh and transfer about
200 mg of Ibuprofen Drug substance into 2 ml of diluent. The mixture is vortex mix
for about 10 minutes and centrifuge for about
30 minutes at 4000 RPM. Resulting solution filter through 0.2 um nylon/mdi filter.
Aliquots of the solution were transferred to UPLC vials for analysis by HPLC.
Procedure: Create sequence as follow and inject the solution according to the
sequence.

S.No. Sample Name No. of Injection
1 Blank (Diluent) 1

2 Standard Solution 6
3 Test Solution 1
4 Standard SolutionBkt 1
Relative retention time for impurity are as follows:

S.No. Name of the Standard/Impurity RRT(minute)
1 Ibuprofen 1.00
2 NDMA 0.57
The retention time of Ibuprofen is about 9.4 minutes and NDMA is about 5.4
minutes.
System Suitability:
• % RSD of six replicate injections of standard solution should not be more than 10.0%.
Calculation (For NDMA):
Calculate the percentage of NDMA impurity in Ibuprofen by using the formula:
AT WS P
Impurity (%w/w) = X X X 100
AS WT 100
Where,
AT: Peak area of known impurity in chromatogram obtained from sample solution.
AS: Average peak area of six replicate injections of NDMA in chromatogram
obtained from standard solution.
WS: Concentration of NDMA WS/RS in standard solution (mg/ml).
WT: Concentration of Ibuprofen in sample solution (mg/ml).
P: Potency of NDMA WS/RS.
Method for NDEA
Buffer: Accurately transfer 1.0 ml Trifluoro acetic acid in 1000 ml MilliQ water, mix
and sonicate. Filtered through 0.45 um filter paper and degas.
Preparation of Mobile phase-A: Buffer (100) %
Preparation of mobile phase B: Acetonitrile (100) %
Diluent preparation: Dilute 10 ml of Perchloric acid in 1000 ml of MilliQwater and
mix. (100)%

Chromatographic conditions:
Column
Flow
Wavelength
Injection Volume
Column oven Temperature
Sample cooler Temperature
Run Time
Elution pattern (Binary/premix)

: ChromCore PFP (250 x 4.6)mm, 5 um or equivalent : 1.0 mL/minutes : 228nm : 200 uL :40°C :5°C : 45 min. Mobile phase A: Mobile phase B (93:07) %v/v.

Preparation of Standard solution (O.Olppm): Accurately weight and transfer about 20 mg each of NDEA working standards into a 200 ml volumetric flask, add about 60 ml of diluent and sonicate for 5 minutes to dissolve. Makeup the volume with diluent and mix (Stock solution-1). Further, dilute 1ml of stock solution-1 to 200ml with diluent and mix (Stock solution-2). Further, dilute 1.0 ml of stock solution-2 to 50 ml with diluent and mix (Standard solution).
Preparation of sample solution (For NDEA): Accurately weigh and transfer about
400 mg of Ibuprofen Drug substance into 2 ml of diluent. The mixture is vortex mix
for about 10 minutes and centrifuge for about 30 minutes at 4000 RPM. Resulting
solution filter through 0.2 um nylon/mdi filter.
Aliquots of the solution were transferred to UPLC vials for analysis by UPLC.
Procedure: Create sequence as follow and inject the solution according to the
sequence.

S.No. Sample Name No. of Injection
1 Blank (Diluent) 1
2 Standard Solution 6
3 Test Solution 1
4 Standard Solution-Bkt 1
Relative retention time for impurity are as follows:

S.No. Name of the Standard/Impurity RRT(minute)
1 Ibuprofen 1.00
2 NDEA 0.75

The retention time of Ibuprofen is about 10.2 minutes and NDEA is about 7.6
minutes.
System Suitability:
• % RSD of six replicate injections of standard solution should not be more than
10.0%.
Calculation (For NDEA): Calculate the percentage of NDEA impurity in Ibuprofen by using the formula:
AT WS P
Impurity (%w/w) = X X X 100
AS WT 100
Where,
AT: Peak area of known impurity in chromatogram obtained from sample solution.
AS: Average peak area of six replicate injections of NDEA in chromatogram
obtained from standard solution.
WS: Concentration of NDEA WS/RS in standard solution (mg/ml).
WT: Concentration of Ibuprofen in sample solution (mg/ml).
P: Potency of NDEA WS/RS.
TESTING FOR NDMA

Drug Substance Maximum Daily Dose (mg/day) Acceptable Intake NDMA (ng/day) Limits of NDMA (ppm)
Ibuprofen 3200 96 0.03
TESTING FOR NDEA

Drug Substance Maximum Daily Dose (mg/day) Acceptable Intake NDEA (ng/day) Limits of NDEA (ppm)
Ibuprofen 3200 26.5 0.008
While the forgoing pages provide a detailed description of the preferred embodiments of the invention, it is being understood that the summary, description and examples are illustrative only of the core of the invention and non-limiting in nature. Furthermore, as many changes can be made to the invention without departing from the scope of the invention, it is intended that all material contained herein shall be interpreted as illustrative of the invention and not in a limiting sense.

We Claim,

1. A robust, industrially viable and cost effective high pressure liquid chromatography (HPLC) method for determining the genotoxic nitrosamine impurities in drug substance or drug product or their combination products comprising the steps of:
a) preparing sample solution comprising drug substance or drug product or their combination products in diluent
b) optionally vortex mix and centrifuge or sonicate
c) filter the solution of step b)
d) performing HPLC using chromatographic conditions:

Input Parameters (range)
Mobile Phase (A:B)% (v/v) 80-95:5-20
Elution mode Isocratic
Column Specification Cig (250X4.6)mm,5um
Wavelength (nm) 225-229
Column Temperature (°C) 35-40
Flow Rate (ml/min) 0.5-1.0
Run Time (min.) 20-65
Linearity (ng/ml) 5-1.1
LOD (ng/ml) 0.5
LOQ (ng/ml) 1.1
Recovery (%) 80-120
e) performing step a) to d) for standards of nitrosamines either individual or in groups of nitrosamines.
f) determining the quantitative values of nitrosamines up to a precision of 1 nanogram level.
2. A robust, industrially viable and cost effective high pressure liquid
chromatography (HPLC) method according to claim 1, wherein said step a)
of preparing the sample solution comprising:
i. weighing the sample comprising drug substance or drug product or
their combination products; ii. adding diluent to the weighed sample followed by mixing.
3. A robust, industrially viable and cost effective high pressure liquid
chromatography (HPLC) method according to claim 2, wherein the diluent

used in step ii) is selected from perchloric acid, MilliQ water and methanol or in combination thereof.
4. A robust, industrially viable and cost effective high pressure liquid chromatography (HPLC) method according to claim 1, wherein in step b) of optionally vortex mix and centrifuge or sonicate the samples prepared is performed for time duration ranging between 10-20 minutes followed by centrifuging or sonicating for 30-60 minutes at rpm ranging between 3000-5000.
5. A robust, industrially viable and cost effective high pressure liquid chromatography (HPLC) method according to claim 1, wherein in step c) the resulting solution is filtered through 0.2um nylon/mdi/PVDF/PTFE/Glass filter.
6. A robust, industrially viable and cost effective high pressure liquid chromatography (HPLC) method according to claim 1, wherein in step d) the injecting volume of the sample applied to the HPLC is ranging between 200-2000ul.
7. A robust, industrially viable and cost effective high pressure liquid chromatography (HPLC) method according to claim 1, wherein in step d), the mobile phase A comprises a mixture of water and 0.1% of an organic acid and mobile phase B is selected from acetonitrile or methanol.
8. A robust, industrially viable and cost effective high pressure liquid chromatography (HPLC) method according to claim 7, wherein the organic acid used in the mixture of mobile phase A is selected from trifluoroacetic acid, formic acid or in combination thereof.
9. A robust, industrially viable and cost effective high pressure liquid chromatography (HPLC) method according to claim 1, wherein the genotoxic nitrosamine impurities consist of N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosoethylisopropylamine (NELPA), N-nitrosodiisopropylamine (NDIPA), N-nitrosodibutylamine (NDBA), N-nitrosomethyl-4-amino-butyric acid (NMBA), in drug substances and drug products or their combination products.