INTRODUCTION:

Psychotropic describes any drug that affects behaviour, mood, thoughts, or perception. The Substance Abuse and Mental Health Services Administration (SAMHSA) National Survey on Drug Use and Health data found that in 2018, 47 million adults over age 18 reported a mental health condition. This is around 1 in 5 adults in the United States. More than 11 million reported serious mental illness. In Some conditions like anxiety, depression, schizophrenia, bipolar disorder, sleep disorders psychotropic drugs are used¹. These medications work by altering neurotransmitters to improve symptoms. Mental health and well-being affect our daily lives. Quetiapine is works by blocking different receptors such as serotonin and dopamine. Quetiapine is used for symptomatic treatment in schizophrenia and acute episodes of bipolar 1 disorder, post-traumatic stress disorder (PTSD)².

Structure:

Fig.1: Chemical structure of quetiapine

IUPAC name: - 2-[2-(4-benzo[b] [1, 4] benzothiazepin-6-ylpiperazin-1-yl) ethoxy] ethanol

Molecular formula: - C₂₁H₂₅N₃O₂S

Molecular weight: - 383.5 gm/Mol

Role of Quetiapine as psychotropic drug. Quetiapine is a treatment for schizophrenia. Quetiapine is the most recently approved atypical antipsychotic. The drug has a fairly broad spectrum of receptor affinity. It demonstrates moderate affinity for 5-HT2A, α1, M1, and H1 receptors, minor affinity for D2 and 5-HT1A receptors, low affinity for 5-HT2C, α2, and D1receptors, and no appreciable affinity for D4 or benzodiazepine receptors. D2 antagonism is similar to that of clozapine.

Quetiapine and has a prescription market size of 30 billion won ($23.7 million). The original product is Alvogen Korea’s Seroquel, with 30 pharmaceutical companies selling generic products. According to industry insiders, the Ministry of Food and Drug Safety recently ordered pharmaceutical companies that manufacture quetiapine-based drugs to submit review data for N-Nitroso-Aryl Piperazine (NNAP), an N-nitrosodimethylamine (NDMA)-based impurity. The ministry’s precautionary measure came after receiving information about NNAP detection in a single quetiapine product. Pharmaceutical companies must submit the evaluation of the possibility of NNAP occurrence in quetiapine-based drugs, interim management standards, and process review data to the drug ministry by May 27. The MFDS also instructed drug makers to submit a representative batch number test results among finished quetiapine products available for sale by July 28. A representative batch number refers to three or more batches nearing the expiration date. According to the ministry, some companies produce fewer than three batch numbers annually, they must submit test and inspection results of all batch numbers. With the addition of quetiapine, the number of drugs that have received ministry investigation for risk of impurities in Korea has increased to 10 -- valsartan, ranitidine³, losartan⁴, nizatidine, metformin⁵, varenicline, irbesartan, montelukast, flavonoids, and quetiapine. Similar to Korean agency, US agency had also given a transition period of two years have been given to pharmaceutical industries for change in process to eliminate impurities. Daily dose of Quetiapine is 800 MDD/day⁶.

(1)

(2)

Quetiapine impurity B N-Nitroso-Aryl Piperazine

Fig. 2: Reaction scheme for synthesis of quetiapine (1) and generation of N-Nitroso-Aryl Piperazine⁷ (2)

As per reaction scheme the product known as 6.2.23 is precursor or n-1 impurity of Quetiapine and has high possibility to present in Quetiapine. As per European pharmacopeia this impurity is known as Quetiapine Impurity B and has IUPAC name as ‘Dibenzo[b,f][1,4]thiazepine-11-yl-piperazine dihydrochloride; 11-Piperazin-1-yl dibenzo[b, f][1,4]thiazepine Dihydrochloride ("DBTP")’. Quetiapine Impurity B has tertiary amine site and in presence of nitrating agent such as nitric oxide (NO) etc. under proper conditions has the potential to produce nitrosamines as a by-product during the manufacturing process such as N-Nitroso-Aryl Piperazine (NNAP), if not properly removed⁸.

Synthesis route of formation of N-Nitroso-Aryl Piperazine (NNAP) in presence of Quetiapine Impurity B.

Control of N-Nitroso-Aryl Piperazine impurity in Quetiapine:

The rate of nitrosation is depend on pH and concentration of amine and nitrite, also basicity of amine is inversely proportional to the rate of nitrosation of 2⁰amine.

Rate= k [Amine] × [nitrite] ².

Any manufacturing process that brings a nitric oxide (NO) source in contact with an amine under acidic conditions has the potential to produce nitrosamines as a by-product during the manufacturing process⁹. N-Nitroso-Aryl Piperazine impurity being a potent Quetiapine specific nitrosamine impurity it has to be controlled in Quetiapine at limit calculated under the guidance given in guideline^10,11. The AIs in nanograms per day and the maximum daily dose (MDD) of the drug substance (DS) from the drug label in milligrams per day can then be used to calculate the maximum nitrosamine concentration limits, in ppm, for individual drug products using the equation below.

Concentration = AI/DS

Since the exposure to nitrosamines is related to the MDD of the drug, different concentrations of nitrosamines (ng/g) may be acceptable for each material evaluated. The acceptable concentration in the material can be calculated using the equation below.

Acceptable nitrosamine content = AI/MDD

AI = acceptable intake of the nitrosamine (ng/day)

MDD = maximum daily dose of the API (g/day)

Table No 1:- Acceptable intake limit (AI) of targeted Nitrosamine (96 ng/day)

Name

Acceptable Concentration (ng/g)

Nitrosamine 1

0.050

(50 mg dose)

0.100

(100 mg dose)

0.250

(250 mg dose)

1.00

(1000 mg dose)

1920

960

384

Quetiapine Daily Dose is 800mg/day. Thus calculating acceptable concentration range of N-Nitroso-Aryl Piperazine impurity.

Calculating

AI = 96ng /day for 1000mg dose

Then ‘X’ ng/day for 800mg dose of Quetiapine,

Therefore X = 96 x 800/1000

X = 76.8ng/day

Thus acceptable concentration range of N-Nitroso-Aryl Piperazine impurity is 76.8ng/day which is 0.096ppm /day for Quetiapine. As Quetiapine is drug administrated to patient with different pathologies and used continuously for extended period. The main objective of this work was to detect and quantify possible N-Nitroso-Aryl Piperazine at 0.177ppm in Quetiapine crude sample by HPLC so as better control of N-Nitroso-Aryl Piperazine is found in Quetiapine Pure sample at level below 0.096ppm. The reason to control in Crude stage which is penultimate stage of Quetiapine final and screening of this N-Nitroso-Aryl Piperazine impurity at this stage guide us the level and multiple various purification process can be incorporated at crude stage so as better control below 0.076ppm can be incorporated in final. To best of our knowledge, only a limited publication is available which report the analysis of N-Nitroso-Aryl Piperazine in Quetiapine API’s or pharmaceutical preparation. A majority of paper reported, utilize LC-MS/MS or GC-HS/MS system for determination of Nitrosamine N-Nitroso-Aryl Piperazine in Quetiapine. No method has been reported for determination of N-Nitroso-Aryl Piperazine in Quetiapine crude stage¹².

MATERIAL AND METHODS:

Reagents, Chemicals,Impurity standard and drug samples:- N-Nitroso-Aryl Piperazine were purchased from Simson Pharma (India). Acetonitrile,Methanol and Ammonium acetate were used from Merck. Milli-Q water was used during study. Ammonia and Sodium Hydroxide was used from Spectro chem and Merck. Sample was purchased from Mackem laboratories. Details of Standards are given below.

Standard details:

Table 2: working standard details and sample details

Standard
Standard Name	Working Standard Number	Retest Date	Make		Purity
N-Nitroso-Aryl Piperazine	CNAQ-WS-001/A	Oct 2023	Simson Pharma		≥98.0%
Quetiapine Impurity B	SZ Q001D02	Nov 2023	Simson Pharma		≥98.0%
Sample
Sample name	Batch number	Make		Purity
Quetiapine	ML02042022EP	Mackem laboratories		≥98.0%

Reagents and chemicals

Table 3: reagent and chemical details

Reagents and Chemicals	Make	Grade	Batch No.
Water	Merck	HPLC Grade	DF2DF72927
Methanol	Merck	HPLC	SJ2SA21443
Acetonitrile	Merck	HPLC	DI2DA21408
Ammonium Acetate	Merck	AR Grade	MI1M711468
Ammonia	Merck	AR

Instrument details

Table 4:- Instrument and Equipment Details

Instrument/Equipment	Supplier/ Manufacturer	Specification/Model	Instrument ID
HPLC	Waters		ENGLP 200
PDA detector	Waters		ENGLP 201
Balance	Mettler Toledo	XS205	ENGLP 71
Balance	Mettler Toledo	AX26DR	ENGLP 02
Sonicator	Bio System	SC-201500620	ENGLP 219
Micro Pipettes	Thermo Scientific	NA	ENGLP 213
			ENGLP 214
			ENGLP 215
Column
Chemistry	Make	Dimension	Column ID
C18	Waters	150mm x 4.6mm x3	ENEM/LC/COL/22/052
C8	YMC Trait	150mm x 4.6mm x3 µ,120°	ENEM/LC/COL/22/093

METHOD DEVELOPMENT:

Experiment 1:

Attempt was made to separate the N-Nitroso-Aryl Piperazine and Quetiapine on basis of physicochemical properties of both compounds on basis of information regarding solubility of Quetiapine available in monograph. It was observed that due to similar in chemical properties, separation on physicochemical properties was very difficult. Hence only option was remaining to bring separation on basis of chromatographic polarity preference.

Experiment 2:

Attempt was made using pharmacopoeial method, but due to high concentration of Quetiapine sample to be inject up to 2,50,000ppm, interference with main peak was observed. Hence new method needs to be developed so as resolution between peak due to Quetiapine and Impurity N-Nitroso-Aryl Piperazine is well resolved from each other where resolution obtained shall be more than 20 at least. Method was developed using Water, Methanol and Acetonitrile as a phase where Ph of water is adjusted to 7.0. C18 column of make Waters having Dimension 150mm x 4.6mm x 5µ. Peak was well resolved. (Resolution more than 20) but due to high concentration unspecified impurity is found to be merging with N-Nitroso-Aryl Piperazine and poor peak shape was observed. On investigation it was observed that unspecified impurity was due to Impurity B of Quetiapine.

Experiment 3:

To bring the resolution between Impurity B of Quetiapine and N-Nitroso-Aryl Piperazine C8 base deactivated column was used of make Thermo Hypersil dimension 150mm x 4.6mm x 3µ. Here, due to selectivity of C8 chemistry, better resolution of more than 1.2 was observed between Impurity B and N-Nitroso-Aryl Piperazine and also N-Nitroso-Aryl Piperazine is well resolved from Quetiapine peak. (Here Resolution more than 20). Also poor peak shape was observed for N-Nitroso-Aryl Piperazine, Poor peak shape was observed along with low response and thus poor S/N ratio.

Experiment 4:

To resolve the issue, QBD were used where Ph range from ph 5.0 to ph 9.0 was used. Better peak shape was observed at pH 9.0 with better peak shape and reasonable S/N ratio. To sustain pH 9.0, YMC Trait column was used where better resolution between Quetiapine Impurity B and N-Nitroso-Aryl Piperazine (Resolution above 2.0) and resolution between Quetiapine and N-Nitroso-Aryl Piperazine above 20.

Fig. 3: Chromatogram of Resolution between Quetiapine and N-Nitroso Aryl Quetiapine and Resolution between Quetiapine Impurity B and N-Nitroso Aryl Quetiapine

Table 5: System Suitability Details

Sr No	Name of element	Retention time (min)	Relative retention time	Resolution (usp)
1	Quetiapine	14.937	1.000	-
2	Quetiapine Impurity B	30.877	2.067	29
3	N-Nitroso Aryl Piperazine Quetiapine	31.593	2.115	2.1

Experiment 5:

Method was optimized for wavelength, and better response was found at wavelength 220nm where area observed in 4-5 digits for 100% concentration. Concentration was finalized and recovery checked for Target level concentration before validation

Finalized Method for Validation:

HPLC chromatographic condition: The HPLC chromatographic condition was achieved on WATERS HPLC using PDA 2998 Detector. HPLC was equipped with YMC TRAIT column (Chemistry C8, 150mm x 4.6mm x 3µ, 120°). 0.01 Molar Ammonium acetate buffer with pH 9.0 along with Acetonitrile and Methanol in gradient ratio was set and employed as Mobile phase with 0.8mL/min flow at column compartment temperature 50°C and sample compartment temperature at 15°C. Analysis was carried at 220nm by injecting 100 µl sample. Mobile phase binary gradient, time, flow rate and curve used for the separation of N-Nitroso aryl Piperazine Quetiapine.

Buffer: 0.01M Ammonium acetate pH 9.0±0.05 with Ammonia

Mobile Phase A: Buffer: Methanol (90:10) (Premixed) Mobile Phase B: (Methanol: Acetonitrile) (65:35) (Premixed)

Table 6: Gradient Programme Details

Sr. No.	Time	Flow	Mobile Phase A%	Mobile Phase B%	Curve
1	0	0.8	95	5	6
2	5	0.8	95	5	6
3	20	0.8	80	20	6
4	30	0.8	60	40	6
5	35	0.8	30	70	6
6	40	0.8	95	5	6
7	50	0.8	95	5	6

Sample was prepared of 25% Concentration (250000 ppm) and respectively impurity standard was prepared of 0.177 ppm and method was optimized and validate.

Method validation:

Developed method was tested on 8 parameters of validation under the criteria laid under ICH Q2 Guidelines they are as follows 1) Specificity 2) Linearity 3) Limit of Quantification 4) Limit of Detection 5) Accuracy 6) Precision 7) Range.

Each parameter was evaluated with system suitability criteria and acceptance criteria. Critical parameter was finalized for resolution was resolution between Quetiapine Impurity B and N-Nitroso Aryl Quetiapine and other criteria to check system precision % RSD of target level shall be NMT 10.0% as per guidelines¹³.

Fig. 4: Chromatogram of System Suitability chromatogram of validation

Specificity:

Definition: Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Specificity was established based on following criteria

1. There should not be any interference to the retention time of N-Nitroso Aryl Quetiapine due to blank solution

2. There should not be any interference to the retention time of N-Nitroso Aryl Quetiapine due to other specified impurities of Quetiapine.

3. There should not be any interference to the retention time of N-Nitroso Aryl Quetiapine due to other unspecified impurities of Quetiapine.

4. Peak due to N-Nitroso Aryl Quetiapine should be spectrally pure.

Specificity test Authenticity was established on spiked sample with possible specified impurities and peak purity characteristic was studied.

(1)

(2)

Fig. 5: Peak Purity Spectra graph (1) and Chromatogram of Spike chromatogram of mix Quetiapine impurities in sample containing Quetiapine Peak and N-Nitroso aryl Quetiapine peak (2).

Linearity:

Definition: The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample¹⁴.

Stock solution was prepared at approximately 177ppm (1770ng/mL) in Buffer: Acetonitrile and from Linearity solution was prepared of concentration level 0.265 ppm,0.177ppm,0.0885ppm,0.0177ppm and 0.00885 ppm by diluting stock solution to middle stock solution of 1.77ppm using micropipette from which, 150% to 5% of target concentration of 0.177ppm was prepared. The testing API were prepared at approximately 2.5 g/10 ml solution in Buffer: Acetonitrile. The Regression analysis data are as follows

Correlation coefficient obtained is ≥ 0.999 over range of 5% to 150% of target concentration of N-Nitroso aryl Piperazine Quetiapine. Response is found to be linear over range of 5% to 150% of target concentration of N-Nitroso aryl Piperazine Quetiapine.

Fig. 6: Linearity cure chart

Limit of Detection and Limit of Quantification.

The sensitivity of this method was assessed by analysing the minimum amount of sample required to obtain a significant result using the Limit of Detection (LOD) and Limit of Quantification (LOQ). The method was validated as per ICH Q2 Validation procedure.

Definition: LOQ is the lowest concentration at which the analyte can not only be reliably detected but at which some predefined goals for bias and imprecision are met. The LOQ may be equivalent to the LOD or it could be at a much higher concentration¹⁵.

Definition: Limit of detection (LOD) and limit of quantitation (LOQ) can be found in the International Conference on Harmonization's (ICH) Q2 Validation of Analytical Procedures. LOD and LOQ are parameters employ to explain the smallest concentration of an analyte that can be reliably measured by an analytical procedure¹⁶.

LOQ and LOD was predicted by linearity sequence using Signal to noise Criteria. Determination of the signal to noise is performed by comparing measured signals from samples with known low concentration of analyte with those of blank samples and by establishing the minimum concentration at which the analyte can be reliably quantified. Typical signal to noise ratio is 10:1. LOQ was observed to be 5% target concentration of N-Nitroso aryl Piperazine Quetiapine were (S/N) ratio was observed to be ‘10.8’. Predicted concentration of LOQ was verified with Accuracy (proximity of a measured value to a real value) and Precision (Proximity of several measurement to one another) by injecting 6 times predicted LOQ concentration and spiking LOQ concentration of N-Nitroso aryl Piperazine quetiapine for 6 times and studying the recovery of all 6 spike sample and evaluating the result under acceptance criteria. LOQ observed to be 5 % of target concentration of N-Nitroso aryl Quetiapine which is 0.00885 ppm (8.85µg/L) which is verified using Accuracy, which was established to be 102.28% which is well within the ICH compliance condition of between 70% to 130% of target LOQ level and Precision compliance is verified as % RSD for six replicate spike recovery data found to be ‘1.80 ’which is well within the ICH limit of not more than 10%.

LOD was established using LOQ value as per ICH guideline using formulae.

LOD = 33.3/100 X LOQ. Here LOD is established as 0.00294 ppm.

Accuracy:

Definition: Accuracy of an analytical method must be reached by the level of conformity between individual results of the method tested in relation to a value as true¹⁷.

Accuracy was carried out on Quetiapine drug substance.

Calculate Accuracy with the formulae

% recovery = [(A spike – A sample) / A standard] * P

Were,

A spike: Area of Peak due to impurity in spike

A Sample: Area of Peak due to impurity in as such sample

A std: Area of Peak due to standard at target level concentration and P: Potency of Impurity.

Accuracy of the developed method was evaluated in sample by establishing spiking of N-Nitroso aryl Quetiapine solution in Quetiapine at 150% to LOQ level of target concentration. Six replicate spiking solution at LOQ and Target level concentration and triplicate spiking solution at 150% level.

Observation of accuracy:

Average Accuracy from 150% of Target level and at Target level concentration was observed well within ICH limit of 80% to 120% for this method and well within ICH limit of 70% to 130% for this method at LOQ level. Thus, this method is content of N-Nitroso aryl Quetiapine is highly accurate.

Precision:

Definition: Precision shall evaluate the proximity of results obtained by test of samples prepared according to the description of the analytical method to be validated. Precision of developed method was checked by preparing solution of spiking the impurity at 150% target level to LOQ level of N-Nitroso aryl piperazine Quetiapine with the Quetiapine drug for six times at Target concentration and LOQ level whereas at 3 times at 150% level at target concentration. The relative standard deviation of the areas at each level at 150%,Target concentration and at LOQ level was found to be ‘1.68’, ‘1.42’and ‘ 1.80’ which is well within the limit prescribed by ICH of not more that 10%. Thus, confirming the developed method is highly precise.

Range:

The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample (including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity. Range of this developed method of N-Nitroso aryl piperazine Quetiapine Content in Quetiapine was established from LOQ level to 150% level of target concentration of 0.177ppm. Range authenticity was established by checking linearity which was observed to be 0.999 over the given range with average accuracy of 100.35 % and precision which was not deviating by average 1.63% RSD. Thus concluded that method is accurate over range of 0.00885ppm to 0.265ppm.

RESULTS AND DISCUSSION:

Based on above result it is concluded that developed HPLC UV method of N-Nitroso aryl piperazine Quetiapine content in Quetiapine is selective specific, linear accurate and precise for identification and quantification of nitrosamines in crude Quetiapine. The detection limit and quantification limit found to be 0.00885 ppm and limit of detection found to be 0.00265 ppm. The described method is highly reliable technique for the identification and quantification of the Nitrosamine at crude stage Quetiapine during quality control screening at intermediate step at so as well control of below 76 ng/g can be shown in final API.

ACKNOWLEDGEMENTS:

The authors are thankful to the professors and management of Gahlot institute of pharmacy, Koparkhairane for supporting this work. The authors are also thankful to the colleagues of analytical division of Enem nostrum, Mumbai for their guidance and cooperation help to carry out this research work.

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Received on 14.05.2023 Revised on 24.11.2024

Accepted on 10.06.2025 Published on 05.09.2025

Available online from September 08, 2025

Research J. Pharmacy and Technology. 2025;18(9):4062-4068.

DOI: 10.52711/0974-360X.2025.00583

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