The effective separation of zuclopenthixol and its impurities was attained utilizing a KNAUER C18 column (250 mm × 4.6 mm, 5µ id), which was maintained at a temperature of 35 °C. The chromatographic conditions included the use of a mobile phase consisting of 0.1 M sodium acetate buffer at pH 4.3 and methanol in 20:80 (v/v) as mobile phase A, while mobile phase B comprised 0.1% formic acid and acetonitrile in proportion of 75:25 (v/v). An isocratic elution mode was employed, with both mobile phase A and B being pumped in equal volumes at 0.8 mL/min flow rate. For detection purposes, a wavelength of 257 nm was chosen based on observations indicating optimal detector response in comparison with other wavelengths for all analytes. Figure 2 presents the chromatograms obtained for placebo, the standard zuclopenthixol solution spiked with impurities, and the pattern for impurity separation and detection. Based on these chromatograms, the specificity of the method for effectively isolating and identifying process-related impurities of zuclopenthixol was confirmed.

Specificity chromatograms in the optimized method. Chromatogram observed while analyzing placebo solution (A) and precision level solution of zuclopenthixol spiked with impurities (0.1%)

The system suitability data demonstrates suitability of system, with the tailing factor measuring below 1.5, resolution between any adjacent eluting analyte peaks exceeding 2.5 and theoretical plates of all analyte peaks exceeding 2500. This outcome underscores the method’s strong selectivity. Sensitivity evaluation was performed by signal-to-noise (s/n) approach, and the outcomes were expressed as LOD and LOQ. The calculated detection limit was 0.009 µg/mL, while the LOQ was determined to be 0.03 µg/mL for both impurity A and impurity B. These results underscore the method’s elevated sensitivity, particularly suitable for quantification of impurities.

The LOQ concentration of impurities, i.e., 0.03 µg/mL was taken as initial concentration for constructing the calibration curve for impurities. The zuclopenthixol standard solution was prepared so the solution have 0.1% of each impurity, and a precise fitting calibration curve was established within the concentration range of 30–180 µg/mL for zuclopenthixol and 0.03–0.18 µg/mL for the impurities investigated in the study. The calibration curve exhibited a strong linear relationship, with notably high correlation coefficients observed for both impurities and standard zuclopenthixol.

The obtained peak area values exhibited %RSD (relative standard deviation) below the threshold for both zuclopenthixol and the two impurities in various precision studies including intraday, interday precision, precision at the LOQ and ruggedness assessments. These findings underscore the method’s commendable precision. To assess accuracy, recovery studies were conducted by spiking concentrations of 50%, 100%, and 150% of the target, equating to 60 µg/mL for zuclopenthixol and 0.09 µg/mL for impurities A and B. % Recovery was calculated for standard and all impurities in each analysis, with %RSD values determined for each spiked level. Incorporating zuclopenthixol and the studied impurities, the achieved % recovery fell within the acceptable range of 98–102%, affirming the accuracy of the method. Additionally, %RSD values at each spiked level remained below 2%, aligning with the acceptable limit and further confirming the method’s accuracy. The summarized outcomes encompassing in validation of proposed method are presented in Table 1.

No notable alteration in the chromatographic response or system suitability was observed when the experiment was conducted with slight deviations in the proposed method conditions. Resolutions between consecutive analytes consistently exceeded 2.0, and tailing factors for all analytes remained within acceptable limits. The variability in zuclopenthixol and impurity estimation stayed below the acceptable threshold of 2, confirming the method’s robustness. The results of the robustness study conducted in the developed method are detailed in Table 2.

Forced degradation studies performed to evaluate the effectiveness of the method for resolution of degradation compounds, and the study was conducted in acid, base, peroxide, thermal and UV light degradation conditions. There is no considerable degradation was noticed in UV light and thermal degradation conditions with % assay of 97.49% and 97.76%, respectively. Among the degradation conditions, high % degradation was noticed in acid degradation study with a % degradation of 8.96%. The chromatogram observed in this study (Fig. 4A) show well-resolved DPs at tR of 2.67 min, 5.20 min and 6.01 min and were named as DP 1, DP 4 and DP 5, respectively. The chromatogram identified in peroxide degradation study (Fig. 4B) clearly resolve two degradation products at tR of 3.98 min and 8.66 min and were designated as DP 3 and DP 6, respectively, with a % degradation of 7.95%. The peak corresponds to impurity B at tR of 1.98 min was also noticed in peroxide degradation chromatogram. The % assay of zuclopenthixol in base degradation was calculated to be 92.37% with mass balance of 99.13%. The chromatogram clearly resolves two DPs at tR of 3.43 min and 8.68 min and these impurities were marked as DP 2 and DP 6, respectively. The results of the peak purity test, as determined by the PDA detector, validated the purity and homogeneity of the zuclopenthixol peak across all examined stress samples. The mass balance for stressed samples fell within the range of 98.93% to 99.71%. These peak purity test outcomes consistently confirmed the homogeneity and purity of the zuclopenthixol peak within the analyzed stress samples. The assay of zuclopenthixol exhibited negligible variation in the presence of impurities, and the peak purity results of the stress samples further substantiate the specificity and capability of the developed method to indicate stability. Comprehensive details are provided in Table 3, while Fig. 3 illustrates representative chromatograms observed during the forced degradation study.

Forced degradation chromatograms of zuclopenthixol. A Acidic stress study chromatogram of zuclopenthixol visualizing DP 1, 4 and 5; B Basic stress study chromatogram of zuclopenthixol visualizing DP 2 and 6; C Peroxide degradation chromatogram of zuclopenthixol visualizing DP 3 and 6

The DPs generated due to stress effect on zuclopenthixol pure drug were characterized through LCMS/MS analysis. The LC conditions optimized in the study were utilized without any change and the mass operating conditions optimized such that the condition produce maximum detection of each mass fragment with very less or no noise. The mass detector operated 3600 V of capillary voltage, 60 V of fragmentor voltage and 65 V of skimmer voltage, 6 L/H flow of drying (nitrogen) gas at 350 °C and 40 Psi of nebulizer gas. The same experiment condition was monitored throughout the analysis and an average of 20–30 scans were conducted. The preliminary test confirms that the positive ion mode was suitable for optimum and maximum detection of all DPs.

The ESI MS spectrum of DP 1 depicted in Fig. 10A, identified at tR of 2.67 min, exhibits a prominent parent ion at m/z 211 (m + 1), suggesting a plausible molecular formula of C14H10S. Additionally, the spectrum displays less abundant product ions at m/z 161 (m + 1) with a molecular formula of C10H8S. Based on the observed fragmentation pattern, the compound is recognized as 9-methylidene-9H-thioxanthene, characterized by a molecular formula of C14H10S and a molecular mass of 210 g/mol. The proposed mass fragmentation pattern of DP 1 is illustrated in Fig. 4.

Mass fragmentation pattern of DP 1

The mass fragmentation spectra of DP 2 (Fig. 10B) reveal a dominant parent ion at m/z 357 (m + 1) when observed under positive ionization mode. Additionally, the spectrum displays fragment ions at m/z 137 (m + 1) resulting from the loss of C12H13ClN. Through accurate mass measurements, the elemental compositions of the molecular ion of DP 2 and all its fragmented ions have been verified. Based on the data obtained, DP 2 has been definitively identified as 1-[(3Z)-3-(2-chloro-9H-thioxanthen-9-ylidene)propyl]piperazine, possessing a molecular formula of C20H21ClN2S. The proposed mass fragmentation pattern of DP 2 is depicted in Fig. 5.

Mass fragmentation pattern of DP 2

The ESI–MS spectrum of DP 3 (presented in Fig. 10C), observed at a retention time of 3.98 min, displays a parent ion at m/z 329 (m + 1) alongside a prominent fragment ion at m/z 244 (m + 1). The parent ion’s molecular formula is identified as C18H17ClN2S, and this corresponds to the fragment ion with a molecular formula of 243 (resulting from the loss of C4H9N2). Further analysis reveals that the compound is characterized as (2E)-2-ethanamine. The structural details of this compound, as well as its associated fragmentation mechanism, are illustrated in Fig. 6.

Mass fragmentation pattern of DP 3

The ESI–MS spectrum, observed at a retention time of 5.20 min (Fig. 10D), exhibits a parent ion at m/z 311, corresponding to the [M + H]+ of DP 4, which forms under acidic stress conditions. Within the spectrum, there are abundant product ions at m/z 160 (m + 1). The integrity of DP 4’s molecular structure is validated through both peak purity testing and CID studies. The collection of these product ions, in conjunction with the parent ion, serves to affirm that DP 4 is indeed N-methyl-N-[3-(9H-thioxanthen-9-ylidene)propyl]ethane-1,2-diamine, having a molecular formula of C19H22N2S. A representation of its structure, alongside the fragmentation mechanism is presented in Fig. 7.

Mass fragmentation pattern of DP 4

The ESI MS spectrum of DP 5 (Fig. 10E) displayed notable product ions at m/z 288 [M + H]+. A significant product ion at m/z 161 possibly resulted from the loss of C6H8S from m/z 284. The elemental compositions of the molecular ion of DP 5 and all its fragmented ions were confirmed through precise mass measurements. Based on these analyses, DP 5 was identified as (3Z)-3-(2-chloro-9H-thioxanthen-9-ylidene)propan-1-amine, with a molecular mass of 287 g/mol and a chemical formula of C16H14ClNS. Figure 8 present its molecular structure and fragmentation mechanism. The characterization of DP 5 in this study aligned with the oxidative degradation product reported by Thummar et al., 2014 [16].

Mass fragmentation pattern of DP 5

Based on the ESI MS spectrum of DP 6 as presented in Fig. 10F, the fragmentation pattern was proposed as presented in Fig. 9, and the compound was finalized as N,N-dimethyl-3-(9H-thioxanthen-9-ylidene)propan-1-amine with molecular formula of C18H19NS and molecular mass of 280 g/mol. This DP 6 was identified in the chromatogram of both base and peroxide stress studies (Fig. 10).

Mass fragmentation pattern of DP 6

Mass spectra of DPs observed in forced degradation study. Mass spectra identified at tR of 2.67 min for DP 1 (A), 3.43 min for DP 2 (B), 3.98 min for DP 3 (C), 5.20 min for DP 4 (D), 6.01 min for DP 5 (E) and 8.68 min for DP 6 (F)

The developed HPLC technique was put into practice to quantify pharmacopeia-defined impurities of zuclopenthixol within a pharmaceutical formulation. The formulation sample underwent direct analysis to assess the impurities present within it. Additionally, a formulation sample spiked with impurities was analyzed to gauge the method’s efficacy in separating and quantifying impurities within the formulation. The chromatogram acquired from the formulation solution spiked with impurities (depicted in Fig. 11) distinctly exhibited peaks corresponding to the impurities under investigation. In contrast, the chromatogram from the un-spiked formulation solution displayed no peaks associated with the studied impurities. This observation implies that the amount of impurity in the sample fell below the detection limit. Consequently, it confirms that the impurity quantity in the sample remained below permissible levels. This substantiates the successful applicability of the proposed method for the accurate quantification of process-related impurities in zuclopenthixol.

Formulation analysis chromatogram of zuclopenthixol. Chromatogram noticed for formulation solution spiked with impurities (A) and with no impurities spiked (B)