INTRODUCTION:

N-nitrosodimethylamine (NDMA) and several N-nitrosamines classified as probable human carcinogens by the US Environmental Protection Agency (US EPA, 1993). NDMA and NDEA are classified by the International Agency for Research on Cancer (IARC) as group 2A carcinogens (probably carcinogens), while N-nitrosoethylethylamine (NMEA) and N-nitrosodibutylamine (NDBA) are classified as 2B (probably carcinogenic). ICH M7 (R1)¹ classifies nitrosamine impurities as Category 1 based on rodent carcinogenicity and mutagenicity data, known to be mutagenic and carcinogenic.

These nitrosamine impurities affect genetic material through mutations such as chromosomal breaks, rearrangements covalent bonding, or insertion into DNA during replication. Exposure to very low levels of nitrosamine impurities can cause these changes in genetic material, which can lead cancer. Therefore, identifying very low levels of nitrosamine impurities in pharmaceuticals is important to ensure public safety.

Nitrosamines, as a group of emerging disinfection of products in purified water particularly when chloramine is adopted as the disinfectant, have recently raised great concerns because of their high carcinogenic potency in comparison to conventional such as trihalomethanes (THMs) and haloacetic acids (HAAs). Until now, six nitrosamines have been detected in purified water, while N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA) are the most frequently detected compounds. NDMA formation mechanisms during chlorination, chloramination, and ozonation processes have been extensively studied by many researchers. Chlorination and Chloramination are the most widely used water disinfection treatments. The process of chloramination is adding chloramines to untreated water to destroy pathogenic microbes and it can also be achieved indirectly by chlorination in the presence of ammonia. The leaching of dimethylamine (DMA) from ion exchange resins has been related with the occurrence of N-nitrosamine in water.

Food and Drug Administration recommends the following acceptance intake (AI) limit in Table 1 for Nitrosamine contamination by NDMA, NMBA, NDEA, NMPA, NDIPA, NIPEA.

Table 1: Limit of Nitrosamine Impurity

Nitrosamine	AI limit (ng/ Day)
NDMA	96.0
NDEA	26.5
NMBA	96.0
NMPA	26.5
NIPEA	26.5
NDIPA	26.5

N-nitrosamines are frequently present in the environment at the ppt (ng/L) level, posing a challenge for accurate quantitative analysis. Therefore, it is necessary to develop an accurate and sensitive analytical detection method. USEPA Method 521 uses ion-Trap for nitrosamine analysis and can be adapted to use GC-MS/MS in El mode to achieve sub-ng/L detection levels in drinking water, as described elsewhere. We demonstrate a sensitive and accurate method for the determination of six common nitrosamines in purified water using tandem liquid chromatography-mass spectrometry. High performance liquid chromatography tandem mass spectrometry (LC-MS/MS)^2,3 has been extensively used to detect the N-nitrosamines in water with high sensitivity. High-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)^4,5 is a powerful and reliable analytical technique for quantitative analysis of compounds at trace levels due to its good linearity, high sensitivity, and selectivity. LC-MS/MS methods have been developed to determine secondary amines in various sample.

The objective of this work was to develop a simple, accurate, sensitive and rapid method of high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)^6–10 for the detection and quantification of traces of NDMA and other N-Nitrosamine water samples. To this end, we have evaluated and optimized several parameters of the mass spectrometer as well as the actual parameters affecting the chromatographic separation, such as the composition of the mobile phase, the mode of elution and the flow rate of the eluent. To the best of our knowledge, this study is the first analytical method for the analysis of trace concentrations of NDMA and other N-nitrosamines in purified water with high extraction recoveries.

MATERIAL AND METHOD:

Chemical and Reagents:

All six N-nitrosamine reference standards impurities were purchased from PS3 labs LLP (Hyderabad, India), including N-Nitroso dimethylamine (NDMA), N-Nitroso diethylamine (NDEA), N-Nitroso Ethyliso-propyl-amine (NEIPA), N-Nitroso diisopropylene (NDIPA), N-Nitroso dibutylamine (NDBA), N-Methyl-4-Amino-butyric acid (NMBA). Formic acid is a ACS grade reagent were purchased from Merck (Darmstadt, Germany), and Methanol were HPLC grade from Finar. Ultra-pure HPLC grade water used in experiment was generated by using a Milli-Q water purification system.

Pharmaceutical Sample:

Purified water samples were provided by a pharmaceutical company.

Preparation of Mobile Phase:

Prepare mobile phase A by adding 1mL of Formic acid to 1000ml of HPLC grade water and mobile phase B by adding 1mL of Formic acid to 1000ml of HPLC grade methanol. The mobile phase was degassed and stored at room temperature for later use.

Preparation of diluent:

Prepare diluent by adding 100mL methanol and 900mL of 0.1% Formic acid solution, mix well and sonicate and store at ambient temperature. It is also used as blank.

Preparation Standard Solution:

Preparation of standard stock solution by weighing 5 mg about of each NDMA, NDEA, NMBA, NDIPA, NEIPA and NDBA standard accurately and transfer separately in 50mL volumetric flask. Adding 5 mL of methanol and Sonicate 4-5min to dissolve and with using diluent make up to the volume and to obtain final concentration of 100 ppm.

Preparation of primary standard stock solution by taking 1mL of each standard stock solution into 100mL volumetric flask and add the 70-80mL of diluent and mix it well and make up the volume with diluent and to obtain final concentration 1ppm. Prepare daily nitrosamine mixtures solution at desired concentrations from stock solutions prior to LC-APCI-MS/MS analysis.

Preparation of sample solution:

Weighing about 1000mg of purified water sample and filter with 0.45µm PVDF filter and filtered purified water sample transferred into HPLC vial for analysis.

Instrumentation condition:

High performance liquid chromatography condition:

The HPLC analysis was carried out on Agilent 1290 infinity II equipped with a quaternary pump. The chromatographic separation was performed using Phenomenex kinetex F5 column (250mm × 4.6mm, 5µ particle size) and the column oven temperature was maintained at 40ºC. The separation of analytes was performed in binary gradient mode of mobile phase were mobile phase A containing 0.1% Formic acid in HPLC grade water and mobile phase B containing 0.1% Formic acid in HPLC grade methanol was used with a constant flow rate of 0.6mL/min. An injection volume of 9.0µL was used for each sample analysis. The elution was performed with a different gradient program as shown in Table 2 and the total run time of 25 min for nitrosamines analysis (Figure no. 1) The needle washed with methanol: HPLC grade water (20:80), after the injection of each sample.

Table 2. The gradient program

Sr. No.	Time (in min)	Mobile Phase A	Mobile Phase B
1	0.01	90	10
2	1.50	90	10
3	7.00	45	55
4	17.00	45	55
5	17.10	10	90
6	21.00	10	90
7	21.10	90	10
8	25.00	90	10

Mass Spectrometry conditions:

The MS-MS detection was used for detection carried out using Agilent 6470 LC/TQ Triple Quadruple instrument. The MS-MS was operated using an atmospheric pressure chemical ionization (APCI) source with positive mode, and detection or quantitation was performed in a multiple reaction monitoring (MRM) mode and it was scanning mode and acquiring mass data for quantification. The MRM parameters, containing MRM transitions, Precursor ions, Product ions, and collision energy and the MRM transition of N-nitrosamine impurities were shown in Table 3.

Table 3. LC-MS/MS MRM Parameters

Analyte	Retention Time (RT) (min)	Quantifier (Q1) (m/z)	Qualifier (Q3) (m/z)	Dwell Time (ms)	Collision Energy (eV)
NDMA	6.8	75.0	58.0	100	18.0
NMBA	9.2	147.1	87.0	100	30.0
NDEA	11.4	103.0	75.0	100	16.0
NEIPA	12.9	117.0	75.4	100	16.0
NDIPA	14.6	131.0	89.0	100	13.0
NDBA	22.8	159.4	57.0	100	22.0

Method validation:

The method validation assay was carried out to examine the system suitability and consistency of the proposed LC-MS/MS method. The characteristic analytical parameters of the present method such as sensitivity in terms of limit of detection (LOD), the limit of quantification (LOQ), recovery (%), linearity w.r.t coefficient of determination (R2), precision, and robustness (%RSD) were calculated as per ICH guideline. The Linearity was constructed by spiking six nitrosamines in the concentration range of 25% - 150%. The calibration curve was drawn by plotting peak area of standard and concentration using a linear least-squares regression method. The linearity of the method is acceptable when the coefficient of determination (R2) was greater than 0.995.

The method’s sensitivity was performed in terms of LOD and LOQ as per ICH validation of analytical procedures. The LOD is the smallest concentration of the analyte, which cannot be quantified but can be detected and LOQ is the smallest concentration of the analyte, which can be quantified with suitable precision and accuracy. The LOD and LOQ were calculated based on the standard deviation and slope of the calibration curve as per ICH Q2 R1 guidelines. Recovery was evaluated at three different concentration levels within the calibration (0.020, 0.040 and 0.080ppm) range of the developed method. Intra-day and inter-day precisions were performed by using six replicates in five consecutive days in terms of %RSD. The blank sample was injected during analysis to know the carry-over effect or error from time to time.

Robustness:

The robustness of the present method was evaluated with deviations of LC conditions such as variation of column temperature (35°C and 40°C) and flow rate of mobile phase (0.55 to 0.65mL/min) in six replicates at the concentration of 0.08ppm.

RESULTS AND DISCUSSION:

Development of the method:

This study was carried out to select an analytical method and develop an optimal method that can separate all six potentially genotoxic nitrosamine impurities in Purified water and perform trace-level quantification. The method development using LC-APCI-MS/MS for nitrosamines was performed. A few columns were screened, such as Phenomenex kinetexF5 and Waters HSS PFP. The mobile phase composition optimization has been performed by taking mobile phases 0.1% Formic acid in HPLC grade water (mobile phase A) and 0.1% formic acid in methanol (mobile phase B) composition was found to give optimum results.

Coelution was observed between Purified water and six nitrosamines’ impurities when tried initially with Phenomenex kinetexF5 250 × 4.6mm, 5µ column. After evaluating to achieve separation between purified water and six nitrosamines’ impurities, Phenomenex kinetex F5250 × 4.6mm, 5µ column could provide the separation along with parallel optimization of gradient method conditions. Both 0.1% Formic acid in HPLC grade water and concluded with 0.1% formic acid in Methanol due to better separation efficiency.

We choose APCI as ionization source in the positive mode because of its good ions selectivity, low susceptibility to matrix components, good value for the determination of nitrosamines, and it was found to be more sensitive to all nitrosamines in the APCI mode. The standard nitrosamine impurities solutions were analyzed in the MRM scan mode to identify quantifier ions and qualifier ions. Selected qualifier ions were used to chose optimal MRM transitions. It was found that the positive ionization type was more sensitive than the negative type at the beginning of the development process due to the polarity of the impurities, resulting in the development process only for positive ionization. The collision energy was optimized by analysing difference between the collision cell voltages to determine the change in MRM sensitivity for all six impurities.

The validation of the developed method was evaluated by using specificity linearity, sensitivity (LOD and LOQ), percent recovery, precision, and robustness.

Method Validation:

The method passes validation because it meets the acceptance criteria. The method has been validated in accordance with the analytical method validation. The method was validated for all important validation parameters (specificity, LOQ, LOD, linearity, precision, percent recovery and robustness).

Specificity:

A single solution of Purified water and mixture of six impurities was prepared at specification level. The spiked purified water solution was then subjected to LC-MS/MS analysis. The obtained results indicates that there is no interference of purified water with all the six impurities N-Nitrosodimethyl amine (NDMA), N-Nitroso diisopropylamino (NDIPA), N-Nitrosodiethyl amine (NDEA), N-NitrosoEthyl Iso propylamine (NEIPA), N-Nitroso-Nmethyl-4-aminobutyric acid (NMBA), and N-Nitroso dibutyl amine (NDBA).

Linearity:

The linearity was well-established from 0.020 to 0.120 ppm for all the six Nitrosamine impurities. The slope, and intercept values and regression coefficient were obtained using least squares linear regression. In between peak the areas and concentrations of impurities, a good correlation was observed in range of 0.9960–0.9999.

Limit of Detection and Limit of Quantification:

The LOD and LOQ of the present method were evaluated by taking 3 times and 10 times the standard deviation at the lowest level of peak concentration divided by the slope of the standard curve, respectively, using Eqs. (a) and (b).As a result, LOD and LOQ of the present method were obtained as0.0066ppm and0.020 ppm, respectively.

LOD = (3.3 × Standard deviation)/Slope (a)

LOQ = (10 × Standard deviation)/Slope (b)

Recovery and Precision:

The recovery (as accuracy) and precision of nitrosamines from the developed method, an amount of Purified water were spiked at different concentrations in triplicates (0.020, 0.040 and 0.080ppm). The percent recovery calculations were done by using the Eq. (c) The intra-day accuracy of nitrosamines for Purified water was found to be in the range of 90.4-107.8%, while precision (%RSD) was found to be in the range of 7.9–9.2%. The inter-day accuracy of nitrosamines for Purified water was in the range of 92.6-108.6%, while precision (%RSD) was 8.2–9.1%. Thus, the recovery and precision of the developed method were in the range of acceptable criteria.

% Recovery = (Amount found in ppm/ Amount added in ppm) × 100 (c)

Robustness:

The robustness of the proposed method was evaluated with deviations of LC conditions such as variation of column temperature (35°C and 40°C) and flow rate of mobile phase (0.55 to 0.65mL/min) in six replicates at the concentration of 0.08ppm.

Application of the developed method for test sample analysis:

The developed method was successfully applied to identify and quantify nitrosamines in Purified water using LC-APCI-MS/MS. The present method successfully showed acceptable sensitivity (LOD and LOQ), linearity, recovery, precision, and robustness for nitrosamines. The developed and validated LC-APCI-MS/MS method was applied to identify and quantify nitrosamines concentration in Purified water. The nitrosamines were found below the detection in purified water.

CONCLUSION:

The developed LC-APCI-MS/MS method is selective, rapid, precise, accurate, and robust for determining six nitrosamines in the Purified water, demonstrating the good performance and specificity in screening and qualification of nitrosamines. The developed method was validated in terms of linearity (0.020-0.120ppm) with coefficient of determination (R2) (0.9990-0.9999), LOD (0.0066ppm), LOQ (0.020ppm), and recovery in the range from 90.4% to 109.6% with good precision. The robustness was evaluated with deviations in LC conditions such as variation in column temperature and variation in the flow rate of the mobile phase. Therefore, the developed and validated method can be applied to screen and quantify nitrosamines in Purified water.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.