METHOD DEVELOPMENT AND VALIDATION OF ANALYTICAL METHOD FOR ESTIMATION OF GENOTOXIC IMPURITIES IN TRAZODONE HCL DRUG SUBSTANCEUSING UFLC-MS/MS (ESI)

METHOD DEVELOPMENT AND VALIDATION OF ANALYTICAL METHOD FOR ESTIMATION OF GENOTOXIC IMPURITIES IN TRAZODONE HCL DRUG SUBSTANCEUSING UFLC-MS/MS (ESI)

Shankrappa Janiwarad, K. R. Ravi Kumar *, P. Venkatesh, B. K. Vanitha Devi, H. Mallesha, B. C. Revanasiddappa, R. S. Chandan, B. S. Ananya and M. R. Hithesh

DSIR Approved Research and Development Centre, Robust Materials Technologies Pvt. Ltd., NGEF Layout, Nagarbhavi, Bangalore, Karnataka, India.

ABSTRACT: Due of their ability to damage DNA, genotoxic impurities (GTIs) in active pharmaceutical substances need strict analytical control. Trace process-related GTIs may be present in trazodone hydrochloride, an antidepressant made using multi-step synthetic processes. Nevertheless, there is currently no approved analytical technique for the simultaneous determination of all significant GTIs related to its production. In order to quantify four process-related GTIs in trazodone hydrochloride at sub-ppb levels, a quick and selective UFLC-MS/MS approach was developed and validated. Acetonitrile-5 mM ammonium acetate buffer (pH 5.0, 70:30 v/v) was used for chromatographic separation on an Avantor ACE C18-PFP column (3 µm, 4.6 × 150 mm) under isocratic conditions. Multiple reaction monitoring was used for detection in positive ESI mode. The approach was validated over 0.060-1.200 ppb. For every impurity, linearity was attained with correlation coefficients (R²) ≥ 0.99. Across three levels, accuracy studies produced recoveries ranging from 79 to 102%. Robustness was verified with intentional changes in flow rate and column temperature, and precision satisfied acceptance standards with %RSD < 15%. When kept in a refrigerator, stock solutions stayed stable for at least three days. All GTIs were found to be below the set regulatory limits when the validated procedure was applied to commercial batches. For routine quality-control monitoring of genotoxic contaminants in trazodone hydrochloride, the established UFLC-MS/MS technique is sensitive, dependable, and appropriate.

Keywords: ICH guidelines, Pharmaceutical quality control, Process related impurities, Mass spectrometry, Drug safety assessment

INTRODUCTION: Trazodone hydrochloride is a triazolopyridine-derived antidepressant.

It was first made available in the 1970s and received US approval in 1978.

As a serotonin antagonist and reuptake inhibitor (SARI), it is often used for treating major depressive disorders and a number of other off-label diseases, such as fibromyalgia, schizophrenia, chronic pain, insomnia, and symptoms connected to substance abuse ^1-6. Its multi-step synthetic procedure can produce process related contaminants, like chemicals with potential genotoxicity, despite being clinically efficacious and well tolerated. Due to their potential carcinogenicity and ability to harm the DNA, genotoxic impurities (GTIs) are considered a major toxicological issue.

ICH, EMA, and USFDA regulatory frameworks mandate strict management of GTIs, usually at very low concentration limits, using strategies like daily dose-based thresholds, the ALARP principle, and the Threshold of Toxicological Concern (TTC) of 1.5 µg/day ^7-12.

Hence, in order to guarantee the safety of active pharmaceutical ingredients (APIs), effective analytical techniques are crucial. Various analytical methods for trazodone-related contaminants have been documented. Using LC-MS/MS with ppb-level sensitivity, Venugopal et al. measured the GTI CCP HCl ¹³.

A stability-indicating HPLC method. Moreover LC-MS/MS techniques for other contaminants (“Compound A” and “Compound F”) have also been reported ^{14, 15}.

However, no published approach quantifies all four primary process-related GTIs generated during trazodone synthesis. By creating and validating a selective UFLC-MS/MS method for the simultaneous quantification of four GTIs- 1-(3-Chlorophenyl) – 4 - (3-chloropropyl) piperazine hydrochloride (CPH), 1-(3-Chloropropyl)-4-(4-chlorophenyl) piperazine (CCP), 1-(3-Chloropropyl)- 4-(3-bromophenyl) piperazine (CBP), and1-(3-Bromopropyl)-4-(3-chlorophenyl) piperazine (BCP) at sub-ppb levels, the study fills this analytical gap and offers a complete tool appropriate for regular quality-control monitoring.

EXPERIMENT:

Chemicals and Reagents: LC–MS grade acetonitrile, methanol, and water were sourced from J.T. Baker (Avantor Performance Materials India Pvt Limited Gujarat, India). LC–MS grade formic acid was obtained from Biosolve (Chimi SARL Netherlands), and glacial acetic acid from (RankemAvantor Performance Materials India Pvt Limited Thane Maharashtra, India). Trazodone hydrochloride API Fig. 1A was obtained from Bajaj Healthcare.

FIG. 1A: MOLECULAR STRUCTURE OF TRAZODONE HCL

The following genotoxic impurities (GTIs) were used:

BCP: 1-(3 - Bromopropyl) – 4 - (3 - chlorophenyl) piperazine Fig. 1B.

FIG. 1B: MOLECULAR STRUCTURE OF 1-(3-BROMOPROPYL)-4-(3-CHLOROPHENYL)-PIPERAZINE

CBP: 1-(3 - Chloropropyl) – 4 - (3-bromophenyl) piperazine Fig. 1C.

FIG. 1C: MOLECULAR STRUCTURE OF 1-(3-CHLOROPROPYL)-4-(3-BROMOPHENYL)-PIPERAZINE

CCP: 1-(3-Chloropropyl) – 4 - (4-chlorophenyl) piperazine Fig. 1D.

FIG. 1D: MOLECULAR STRUCTURE OF 1-(3-CHLOROPROPYL)-4-(4- CHLOROPHENYL)-PIPERAZINE

CPH: 1-(3-Chlorophenyl) – 4 - (3-chloropropyl) piperazine hydrochloride Fig. 1E.

FIG. 1E: MOLECULAR STRUCTURE OF 1-(3-CHLOROPHENYL)-4-(3-CHLOROPROPYL) PIPERAZINE HYDROCHLORIDE

Preparation of Standard Solution: 1mg/ mL of each genotoxic impurity was prepared by dissolving 10mg of impurity in 10 mL LC-MS grade methanol separately. Acetonitrile: 5mM ammonium Acetate pH 5 (70:30); pH of 5mM ammonium Acetate was adjusted with 0.1% glacial acetic acid; was used as the diluent for the preparation of working standard solutions and sample solutions. The individual stock solutions of the impurities mixed to get the system suitability solution with concentration of 0.6 ppb.

Sample Solution Preparation: Trazodone Hydrochloride Sample was prepared at concentration of 1mg/mL (w/v) using the Diluent and injected on the chromatography.

Instrument and Analytical Parameters:

Mass Spectrometry: Analysis was carried out on a Shimadzu LC–MS/MS 8045 triple quadrupole system equipped with a SIL-40C×3 autosampler and LC-40B×3 solvent delivery modules, operating in positive Electrospray Ionization (ESI+) mode. Quantitation was carried out using Multiple Reaction Monitoring (MRM), with instrument parameters optimized to yield the most abundant and selective precursor-to-product ion transitions. Each analyte was identified based on its mass-to-charge (m/z) ratio and characteristic fragmentation pattern, as illustrated in Fig. 2A (BCP), Fig. 2B (CBP), Fig. 2C (CCP), and Fig. 2D (CPH). The complete set of optimized source, ionization, and compound-dependent parameters used for MRM acquisition is summarized in Table 1.

FIG. 2A: MASS SPECTRUM OF PRECURSOR ION FOR 1-(3-BROMOPROPYL)-4-(3-CHLOROPHENYL)-PIPERAZINE (BCP) OBTAINED UNDER OPTIMIZED UFLC–MS/MS CONDITIONS IN POSITIVE ESI MODE

FIG. 2B: MASS SPECTRUM OF PRECURSOR ION FOR 1-(3-CHLOROPROPYL)-4-(3-BROMOPHENYL)-PIPERAZINE (CBP) OBTAINED UNDER OPTIMIZED UFLC–MS/MS CONDITIONS IN POSITIVE ESI MODE.

FIG. 2C: MASS SPECTRUM OF PRECURSOR ION FOR 1-(3-CHLOROPROPYL)-4-(4-CHLOROPHENYL)-PIPERAZINE (CCP) OBTAINED UNDER OPTIMIZED UFLC–MS/MS CONDITIONS IN POSITIVE ESI MODE

FIG. 2D: MASS SPECTRUM OF PRECURSOR ION FOR 1-(3-CHLOROPHENYL)-4-(3-CHLOROPROPYL)-PIPERAZINE (CPH) OBTAINED UNDER OPTIMIZED UFLC–MS/MS CONDITIONS IN POSITIVE ESI MODE

TABLE 1: MASS SPECTROMETRIC AND UFLC CONDITIONS

UFLC Conditions: The chromatographic separation was achieved on Avantor ACE (C-18 PFP, 3µm, 4.6*150mm). Acetonitrile: water containing 5mM ammonium Acetate at pH 5 (70:30 v/v) as mobile phase. The UFLC conditions are depicted in Table 1.

RESULTS AND DISCUSSION:

Method Development: A systematic literature search was performed for the development of this method. The intention was to develop method for the estimation of Genotoxic Impurities due to which, the reported method for estimation of Trazodone Hydrochloride samples was selected for the preliminary trials of the method development ¹³. The experimental conditions were selected by considering the similar physicochemical properties of Trazodone Hydrochloride and genotoxic impurities. The Mass spectrometric optimization was done by performing the auto optimization technique for Genotoxic impurities. Initial trials were found to be promising. Different columns and combination of diluents were tested to improve the peak shape and the retention time of the impurity. During method development, the matrix showed maximum absorbance at 254nm on the Photo Diode Array (PDA) and retention time of 3.490 minutes. According to the method, the eluent was diverted from the column to the drain for the first 4 minutes in order to avoid contamination of the mass spectrometry from higher concentration matrix substances and drugs which are non-targeted analytes. In our optimized method, the eluent was directed to the mass spectrometer after 4 minutes, where the genotoxic impurity eluted, indicating method selectivity as depicted in Fig. 3, based on the PDA data.

FIG. 3A: CHROMATOGRAMS OF THE UNSPIKED SAMPLE SOLUTION

FIG. 3B: CHROMATOGRAMS OF THE SPIKED SAMPLE SOLUTION

Short analysis duration (7.00 minutes) allows the method to be applied for routine quality control tests within a limited time period. For impurities CPH the retention time was found at 5.59 and for CCP at 5.56. It was found that along with these two impurities there was adjacent unknow peak identified at 5.85 in both analytes as shown in Fig. 3A and 3B.  These peaks were also observed in the standard solutions and sample solutions except blank. The unknown peaks are found to be well separated from the analyte of interest and have no impact on the recovery of the analytes. For other two impurities i.e. CBP and BCP no unknown peaks observed.

Method Validation: The developed method was validated for the estimation of Genotoxic Impurities in Trazodone Hydrochloride. The method was validatedover the range of 0.060ppb to 1.200ppb for all the impurities, since the limit is same for all the impurities and was evaluated by employing linearity, sensitivity (LOD and LOQ), percent recovery, intra- and inter-day precision, and robustness.

The stock solution was also established for 3 days at refrigerated condition.  The results are depicted in Table 2.

TABLE 2: VALIDATION SUMMARY RESULTS

System Suitability: The system suitability was evaluated by using six replicate injections of the standard solution at 0.600 ppb for all impurities, equivalent to the limit with respect to sample.

Sensitivity (LOD and LOQ): Based on the recommended acceptable daily intake, LOD and LOQ were set at 7.5% and 10% of the daily acceptable limit respectively. Accordingly, the LOD was established at 0.047ppb and LOQ at 0.0625ppb. LOD corresponded to a signal-to-noise(S/N) ratio greater than 3 and LOQ corresponded to a S/N ratio more than 10 with a % RSD for 6 replicate injections of LOQ not exceeding 20%.

Carryover: Carryover test was established to test and measure the unintended transfer of the analyte from one test solution to another. Established by injecting lower and standard solution at 200% level of limit along with inter dispersed blank solutions in the sequence. No interference should be observed at the retention time of analyte in blank solutions, if observed it should be less than 20% of High concentration Standard solution.

Selectivity: The selectivity was established by analysing the sample and spiked sample at limit level to verify the interference at the retention time of the analyte.

The chromatograms of blank, unspiked, and spiked samples Fig. 3A and Fig. 3B confirmed the absence of interfering peaks at the retention times of BCP, CBP, CCP, and CPH.

The trazodone API peak was also well resolved. The unknown peak at 5.85 min remained baseline separated and did not affect analyte determination

Linearity: A 6-point calibration curve (0.060, 0.150, 0.300, 0.600, 0.900, 1.200ppb) was prepared to establish the linearity in a concentration range of 0.060ppb to 1.200ppb.

The coefficient of determination (R²) for the Genotoxic Impurities was determined to be greater than 0.99. Fig. 4A (BCP), Fig. 4B (CBP), Fig. 4C (CCP), Fig. 4D (CPH). Calibration plots are shown in Fig. 4A-4D, each corresponding to one impurity.

FIG. 4A: CALIBRATION CURVE FOR 1-(3-BROMOPROPYL)-4-(3-CHLOROPHENYL)-PIPERAZINE (BCP)

FIG. 4B: CALIBRATION CURVE FOR 1-(3-CHLOROPROPYL)-4-(3-BROMOPHENYL)-PIPERAZINE (CBP)

FIG. 4C: CALIBRATION CURVE FOR 1-(3-CHLOROPROPYL)-4-(4-CHLOROPHENYL)-PIPERAZINE (CCP)

FIG. 4D: CALIBRATION CURVE FOR 1-(3-CHLOROPHENYL)-4-(3-CHLOROPROPYL)-PIPERAZINE (CPH)

Accuracy and Precision: Recovery for the method was evaluated by spiking the sample at 3 different levels (Lower level- 0.150ppb limit-level-0.600ppb and Upper level-0.900ppb) in triplicate. Percent recovery was calculated using the equation:

% Recovery = Concentration obtained / concentration Spiked x 100

The accuracy of the method was confirmed as the recovery of the impurity at each level was within 100±20 %. The precision was established by spiking the sample at the daily acceptable limit (0.625 ppm) in 6 preparations. The observed %RSD for precision was found to be less than 10.0%. Intermediate precision was assessed by a second scientist on a different day using the same instrument. The obtained results from the intermediate precision experiment indicated that the % RSD value of the six samples of genotoxic impurities was less than 15.0 % and the combined % RSD of the 12 solutions was less than 20.0 %.

Robustness: Robustness indicates the ability of the method to remain unaffected by small variations in the method parameters, such as flow rate (with 10% variation in set flow i.e.0.720 -0.880 mL/min) and column temperature (with 5% variation of set value i.e. 35^oC-45^oC) by injecting 6 replicate injections of the standard solution at 0.600ppb. The %RSD for the analyte remained within the acceptance criteria i.e. NMT 15%.

Stock Solution Stability: Stock solutions of all impurity standards established by analysing the fresh standard solutions against the standard solutions stored at refrigerated temperature for specific period of time. Stock solution stability was established for 3 days at refrigerated temperature.

FIG. 5: CHROMATOGRAMS OF COMMERCIAL TRAZODONE HYDROCHLORIDE SAMPLES.

Sample Analysis: The validated method was successfully applied for the estimation of Genotoxic Impurities in commercial samples of Trazodone Hydrochloride. System suitability and bracketing standards were consistent with the acceptance criteria. The Genotoxic Impurities were below the acceptance criteria. The chromatograms of each sample is depicted in Fig. 5.

CONCLUSION: The four process-related genotoxic contaminants in trazodone hydrochloride can be monitored at sub-ppb levels using the extremely sensitive and selective UFLC–MS/MS technique. Even in the presence of small degradant peaks, the method consistently showed outstanding analytical performance, including excellent linearity, precision, accuracy, and resilience, guaranteeing dependable quantification. Standard solutions continued to be appropriate for multi-day usage, enabling regular laboratory processes, according to stability evaluations. Method’s relevance for quality assessment was highlighted by the analysis of commercial API batches. This confirmed that all contaminants were present well below regulatory limits. The technology ensures adherence to international requirements for genotoxic impurity monitoring and is ideally suited for routine quality-control contexts due to its quick runtime, high repeatability, and capacity to identify impurities at trace concentrations.

ACKNOWLEDGEMENTS: The authors acknowledge Robust Materials Technologies Pvt Ltd., J.S.S College of Pharmacy and VR Unnati Health and Education Trust for access to the resources.

Data Availability: The data supporting this study's findings are available from the corresponding author, upon reasonable request.

CRediT Authorship Contribution Statement: Shankrappa Janiwarad: Writing – original draft, Methodology, Investigation, Conceptualization, Data curation. Dr. K.R. Ravi Kumar, Vanitha Devi B. K., Dr. Mallesha. H: Facilitating the instrumentation, materials and facility to carry out the development and validation. Dr. Venkatesh P, Dr. B.C. Revanasiddappa, Dr. Chandan R S: Visualization, Methodology, Review and Editing, Supervision, Conceptualization, Data curation. Ananya B S, Hithesh M.R: Formal Analysis, Literature search, Writing Conceptualization.

CONFLICT OF INTEREST: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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    How to cite this article:

    Janiwarad S, Kumar KRR, Venkatesh P, Devi BKV, Mallesha H, Revanasiddappa BC, Chandan RS, Ananya BS and Hithesh MR: Method development and validation of analytical method for estimation of genotoxic impurities in trazodone HCl drug substance using UFLC-MS/MS (ESI). Int J Pharm Sci & Res 2026; 17(4): 1178-89. doi: 10.13040/IJPSR.0975-8232.17(4).1178-89.

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