Ultra‐Sensitive and Validated method for Trace Quantification of Carcinogenic Benzene in Cetirizine Dihydrochloride

ABSTRACT

Benzene (BENZ) is classified as a Class 1 residual solvent according to ICH Q3C guidelines due to its established classification as a Group 1 human carcinogen. During the chemical production of Cetirizine Dihydrochloride (CTZ) API, Cetirizine Dihydrochloride tablet (CTZT), Levocetirizine Dihydrochloride (LCTZ) API, and Levocetirizine Dihydrochloride tablet (LCTZT), BENZ can appear as a residual process impurity with carcinogenic risk; development of a highly sensitive quantification method is a critical requirement for pharmaceutical quality control. This study focused on developing and validating a method for detecting trace‐level BENZ in CTZ and LCTZ drug substance and drug product. The method demonstrated exceptional sensitivity, with a least detectable concentration of 0.04 ppm and a quantifiable concentration of 0.12 ppm, significantly lower than the ICH‐mandated safety limit of 2 ppm. Linearity and accuracy studies yielded an outstanding percentage of samples spiked BEN in the drug substance and drug product of CTZ and LCTZ and found within acceptance limit, approving the method's validity in presence of the drug atmosphere. Conventional headspace GC‐FID/GC‐MS are established techniques for benzene analysis; the proposed HPLC method offers a simpler, cost‐effective, and validated method, making it well suited for routine quality control laboratories where GC facilities may not be readily available.

1. introduction

In pharmaceutical synthesis, BENZ serves primarily as a versatile building block and chemical intermediate rather than a process solvent, forming the structural core of a significant majority of small‐molecule drugs. Due to its potent carcinogenic properties and environmental risk, the pharmaceutical industry strictly regulates BENZ as a Class 1 residual solvent (ICH ²⁰²¹). Based on an IARC study on cancer, BENZ is classified as a Group‐1 carcinogenic compound, as there is definitive evidence that it causes cancer in humans, particularly blood‐related malignancies (American cancer society ²⁰²³; IARC ²⁰¹⁸). Under international ICH Q3C guidelines and USP standards, its use is heavily discouraged and restricted to a maximum concentration of 2 parts per million (ppm) in finished products (Snodin 2025). Consequently, any concentration exceeding the established permitted daily exposure (PDE) is treated as a critical safety concern that can trigger immediate product recalls to safeguard patient health (US FDA ²⁰²⁵).

CTZ is a selective H1 antagonist drug substance used in various allergic conditions such as hay fever, dermatitis, and chronic urticaria etc. It works by blocking histamine released by the body during allergic condition with the chemical name (±)‐[2‐[4‐[(4‐chlorophenyl)phenylmethyl]‐1‐piperazinyl]ethoxy] acetic acid dihydrochloride. It is commonly known as Zyrtec. The structural formula for CTZ and BENZ are stated in Figure 1, and possible potential sources of BENZ contamination in CTZ/LCTZ are shown in Figure 2 (Campoli‐Richards et al. ¹⁹⁹⁰; Curran et al. ²⁰⁰⁴).

FIGURE 1.

Structure of CTZ and BENZ.

FIGURE 2.

Possible potential sources and benzene contamination CTZ/LCTZ.

To establish a robust BENZ control strategy in pharmaceutical manufacturing, companies must implement a multi‐layered approach that spans from raw material procurement to final product testing. The primary strategy involves the qualification of reagents and solvents, which may harbor trace BENZ impurities. Compliance is finally verified at the API stage to ensure the finished drug product does not exceed the PDE of 0.02 mg/day, thereby safeguarding patient safety and preventing product recalls.

Comprehensive research on the development and evaluation of genetic compound present in CTZ and LCTZ drug substance and drug product remains limited. Limited research exists on the validation and analysis of potentially genotoxic impurities in CTZ (Gharat et al. 2025; Patel et al. ²⁰²⁵; Gharat et al. ²⁰²⁶). A heart‐cutting liquid chromatography–mass spectrometry method was developed to simultaneous analysis of seven process‐related impurities using ESI‐MS equipped with a single quadrupole in CTZ tablets (Rudaz et al. 2003). The optimization and validation of reverse‐phase liquid chromatographic method for the estimation of cetirizine mannitol ester impurity in cetirizine hydrochloride chewable tablet are available (Sharma et al. 2011). Also, Ion‐pair‐based fibrous material using liquid‐phase separation combined with HPLC for the simultaneous determination of urinary BENZ, toluene, and styrene metabolites is available (Bahrami et al. 2018). The simultaneous, trace‐level determination of BENZ and 1, 2‐dichloroethane by GC‐HS/GC‐MS in several pharmaceutical drug substances is available with LOD 0.2 and 0.6 ppm, respectively (Sultana and Nagarajan 2019). The presence of BENZ in food items by purging and gas chromatography employed with a mass detector was used (Barshick et al. 1995). BENZ and a number of aromatics were determined in commercially made gasolines using gas chromatography and neutron mass resonance techniques (Singh et al. 2003). Rapid gas chromatography method with liquid injection for the trace‐level determination of BENZ, and other solvents in commercial gasoline samples is available (Miranda et al. 2017). Quantifiable analysis of trace‐level BENZ and related solvents in cellulose acetate tow using heart‐cutting multicomponent GCHS instrument was reported (Ji et al. 2016). The presence of a carcinogenic substance like BENZ in CTZ may pose potential health risks. And so, it is recommended by regulation to have a well‐established analytical method to check BENZ in CTZ in order to ensure safety of the drug. This method provides high sensitivity and enhanced selectivity by elimination of long process for sample preparation or derivatization that determines BENZ in drug substance (US FDA ²⁰²⁴; Jan et al. ²⁰²⁵; El‐Abasawy et al. ²⁰²³; Ucer et al. ²⁰²⁵). Although headspace GC‐based techniques are the conventional approach for benzene analysis, their implementation requires dedicated headspace instrumentation and optimized thermal equilibration conditions. The present HPLC method simplifies the analytical procedure through simple liquid‐phase sample preparation while providing validated analytical performance. The developed HPLC method is regulatory compliant with respect to analytical method validation under the rules described in Q2R2 guidelines (ICH ²⁰²³).

2. Reagents and Methodology

2.1. Standards, Reagents, and Drug Samples

Drug substance CTZ and LCTZ used were provided by pharmaceutical manufacturing Indoco Research Centre from Navi Mumbai unit situated in Maharashtra state of India. Drug products of CTZ and LCTZ were purchased commercially available makes such as Dr. Roddy's, Cipla, Hetrohealthcare, Mankind Pharma, Leeford Pharma, Systopic laboratories and Sun Pharma. Standards such as BENZ and Acetonitrile are procured from Spectrochem Private limited, Mumbai, Maharashtra, India. Standard of Ortho phosphoric acid purchased from Merck Chemicals, U.S.A were also used.

2.2. Instrumentation

The standard and sample preparations were weighed using a Sartorius analytical balance. Chromatographic analysis was performed on a Waters e2695 HPLC system (USA) equipped with a 2489 UV/Vis detector and a 2998 photodiode array (PDA) detector. Instrument control, data acquisition, and processing were carried out using Empower software (version 3.6.1.2).

2.3. Methodology

Chromatographic parameters optimized for the HPLC method, such as column temperature, flow rate, injection volume, wavelength of detection, run time, and gradient composition are tabulated below in Table 1.

TABLE 1.

(A) Chromatographic condition (B) Gradient time‐composition program.

(A) Chromatographic condition
Parameter	Condition
Solution‐1in percent	0.1% Ortho phosphoric acid
Solution‐2 in percent	Acetonitrile
Ratio of the mobile phase	Binary gradient
Blank	Diluent
Diluent	Water
Column temperature	35°C
Column	Inertsil ODS 3, 5 um,4.6 mm × 250 mm
Injection volume	30.0 microlitre per injection
Flow in volume	1.00 mL
Analysis time (hh:mm:ss)	00:35:00
Wavelength	210 nm
Retention time of CTZ/LCTZ	2.611
Retention time of BENZ (analyte)	About 19.5 min
Tailing factor of BENZ (analyte)	1.05
Theoretical plate count of BENZ (analyte)	15796.01
Purity angle of BENZ (analyte)	2.14
Purity threshold of BENZ (analyte)	2.78
S/N ratio (analyte) in LOD solution	5.75
S/N ratio (analyte) in LOQ solution	20.79
S/N ratio (analyte) in Standard solution	47.31
%RSD for RT of BENZ in six replicate	0.03
%RSD for area of BENZ in six replicate	0.06

(B) Gradient time‐composition program
Time (minutes)	Solution‐1 (in percent)	Solution‐2 (in percent)
0	75.0	25.0
5	75.0	25.0
10	60.0	40.0
12	50.0	50.0
18	50.0	50.0
22	50.0	50.0
25	25.0	75.0
30	25.0	75.0
35	25.0	75.0

2.4. Analytical Solutions

2.4.1. BENZ Stock Solution (1000 μg/mL)

Accurately weighed about 100 mg of BENZ standard into 100 ml volumetric flask, shake well, and dilute with diluent.

2.4.2. BENZ Limit Level Standard (0.4 μg/mL)

Transfer 0.1 mL of BENZ Stock Solution to a 100 mL volumetric flask, shake well, and dilute with diluent.

2.4.3. CTZ/LCTZ Drug Substance Sample Solution

CTZ drug samples are prepared using diluent to make a concentration of 200 mg/mL and the solution was sonicated for 2 min, shaken well. This solution was then filtered out using a 0.45 micron syringe filter and analyzed.

2.4.4. CTZ/LCTZ Drug Product Sample Solution

An accurately weighed quantity of finely powdered CTZ/LCTZ tablets, equivalent to 200 mg of CTZ, was transferred into a separating funnel. The tablet powder was prepared by grinding the tablets using a mortar and pestle. Subsequently, 20 mL of water and 15 mL of methylene dichloride (MDC) were added, and the mixture was shaken thoroughly. The organic (MDC) layer was collected in a 100 mL beaker. The extraction was repeated with an additional 15 mL of MDC, and the organic layer was combined with the first extract. The combined extract was evaporated to dryness at room temperature under a gentle stream of nitrogen using a nitrogen evaporator. The remainder was reconstituted by gradually adding 1.0 mL of acetonitrile with thorough mixing. The resulting solution was collected as the final sample solution and subjected to HPLC analysis (Nakka, Muchakayala, and Manabolu Surya ²⁰²⁴; Nakka, Katari, Babu, and Muchakayala ²⁰²³).

3. Result Summary and Dialogue

Initially, multiple trials are carried out to develop HPLC test procedure. Different columns and mobile phase were applied; nonetheless, unsuccessful outcomes were received, as described in Subsection 3.1. Further, the finest response was accomplished for BENZ peak using acidic buffer with binary composition and Inertsil ODS‐3 column as stationary phase, as described in Subsection 3.2.

3.1. Optimization of HPLC Conditions

At the beginning optimization experiments on HPLC technique for BENZ analysis do not yield good results due to broader peak shape of BENZ eluting with gradient baseline drift. Several columns, including Hypersil BDS C8 (100 mm × 3 mm × 3 μm), Hypersil Gold C18 (250 mm × 4.6 mm × 5 μm), and Inertsil ODS pH‐3 (100 mm × 4.6 mm × 3 μm) were experienced but were unable to get appropriate response for the analyte. At the same time, various buffer solutions containing potassium dihydrogen phosphate (KH₂PO₄) buffer at pH 7.0 and 3.0, further buffer K₂HPO₄ with pH 7.0 and 3.0 with Acetonitrile in the ratio 7:3, show non‐symmetrical peak shape of BENZ as shown in Figure S1.

3.2. Confirmation of HPLC Method

The BENZ peak of good symmetry was obtained on Inertsil columns with dimension of Octadecylsilane‐3 of length 25 cm, internal diameter of 4.6 mm and particle size of 5 μm. Response of analyte was achieved due to mixed ratio of acidic Solution 1 and acetonitrile solvent as Solution 2. During analysis, the PDA detector was also employed, which shows BENZ response at maxima wavelength 210 nm, as well illustrated in Figures 3 and 4 respectively, and was successively applied for analytical method validation.

FIGURE 3.

HPLC chromatogram showing proper peak elution of BENZ with linear response.

FIGURE 4.

HPLC spectrum index showing maxima at 210 nm wavelength using PDA detector.

3.3. Analytical Method Validation

To confirm compliance with respect to ICH Q2R2 guideline (Matos et al. 2015), the analytical methodology involved an all‐inclusive validation (Moorthy et al. 2022 ; Kavitapu et al. ²⁰²³). The parameters assessed were Specificity, Accuracy (Recovery), LOD, LOQ, Precision, Linearity, Sample holding stability, and Robustness (Kumar et al. 2011; Mangukiya et al. ²⁰²³).

3.3.1. System Suitability

As recommended in USP General Chapter < 621 >, system suitability was assessed before analysis by injecting six replicate preparations (n = 6) of a 0.4 μg/mL benzene standard solution. Chromatographic performance was evaluated using the %RSD of benzene peak area and retention time (≤ 2.0%), theoretical plates (> 5000), USP tailing factor (≤ 2.0), and signal‐to‐noise ratio (≥ 10). Peak purity for the analyte (purity angle should be less than purity threshold). The system fulfilled all predefined suitability requirements, demonstrating acceptable chromatographic performance for trace benzene determination. The system suitability results are summarized in Table 3, and a representative chromatogram is shown in Figures S2 and S3 (Nakka, Muchakayala, and Surya ²⁰²⁵; Nakka, Katari, Muchakayala, et al. ²⁰²³).

TABLE 3.

(A) Repeatability and Ruggedness, (B) Repeatability at LOQ level, (C) Repeatability of LOD, and (D) Spiking study.

(A) Repeatability and ruggedness					(B) Repeatability at LOQ level
Criteria: ≤ 15%RELSTDDEV					Criteria: ≤ 15%RELSTDDEV
Analysis replicates	RT in time	Precision BENZ in ppm	Ruggedness BENZ in ppm		Analysis level	%Relative deviation
			Inter variability
			Equipment	Analyst
1	19.39	2.03	2.00	1.95	Limit of quantitation	2.18
2	19.38	2.00	1.99	1.97
3	19.37	1.99	2.00	1.92
4	19.36	2.01	2.00	1.98
5	19.38	2.00	2.00	2.02	(C) Repeatability of LOD
6	19.38	1.99	1.99	0.99	Criteria: BENZ peak should be present
Mean	19.38	2.000	2.000	1.971
STDDEV	0.010	0.014	0.006	0.034	Injection‐1	641
%RELSTDDEV	0.05	0.72	0.31	1.73	Injection‐2	634
%Cumulative RSD			0.56	1.52	Injection‐3	657
(D) Spiking study
Criteria: Percentage recovery results ranging between 80 and 120
Spiked in API/tablet	Sr No	Quantifiable level		100% level	150% level
Spiked in CTZ API	1	104.20		99.35	98.63
	2	96.98		98.98	99.11
	3	101.73		99.20	99.02
	Mean	100.97		99.18	98.92
	STDDEV	3.67		0.18	0.26
	%RELSTDDEV	3.63		0.19	0.26
Spiked in CTZ tablet	1	113.27		97.61	84.99
	2	111.31		98.48	84.78
	3	115.10		98.53	84.78
	Mean	113.22		98.21	84.85
	STDDEV	1.89		0.52	0.12
	%RELSTDDEV	1.67		0.53	0.14
Spiked in LCTZ tablet	1	101.52		97.51	83.00
	2	100.96		94.98	82.79
	3	101.92		96.22	83.04
	Mean	101.47		96.24	82.95
	STDDEV	0.48		1.27	0.13
	%RELSTDDEV	0.48		1.32	0.16

3.3.2. Specificity

Specificity termed as the desired analyte should be free from other substances in the given analytical method. The method is said to be specific and was confirmed with HPLC technique using a PDA detector. HPLC graphs were recorded for the BENZ standard, the test solution, and a spiked test sample. These results showed that the BENZ peak had a good separation profile, with no interference observed in spiked test sample from other components in the blank, Placebo or test solutions (CTZ and LCTZ API, CTZ tablet 10 mg, LCTZ tablet 10 and 5 mg) at the BENZ's retention time. Results are recorded as tabulated in Table 2 and Figure 5.

TABLE 2.

Detection of BENZ.

Chromatogram	Detection (at minute)
BENZ standard	19.555
Diluent as blank	Not present
BENZ spiked in CTZ	19.626
CTZ sample	Not present
LCTZ sample	Not present
CTZ tablet (10 mg)	Not present
LCTZ tablet (10 mg)	Not present
LCTZ tablet (5 mg)	Not present
Placebo of CTZ tablet (10 mg)	Not present
Placebo of LCTZ tablet (10 and 5 mg)	Not present

FIGURE 5.

(A) Standard solution, (B) Spiked test solution, (C) Test solution, and (D) Blank.

Above HPLC graphs conclude, diluent and CTZ samples used in analysis were free from interference at the BENZ's detection time in the spiked CTZ samples. Hence, HPLC methodology proves the specificity of the analysis.

3.3.3. Determination of Detection and Quantifiable Levels

The minimum detection and quantitation levels have been obtained from the regression slope of the linearity study and are 0.04 and 0.1 ppm with respect to sample concentration respectively. The linear regression (R ²) plotted and stated in Figure 6A.

FIGURE 6.

(A) Detectable and quantifiable levels of BENZ (B) Linear study of BENZ.

Calculated limit of detection and limit of quantitation by using the following formula:

$$\text{Limit of Detection}=\frac{3.3\mathrm{\sigma }}{\mathrm{S}},\text{Limit of Quantitation}\frac{10\mathrm{\sigma }}{\mathrm{S}}$$

where σ = Standard deviation of the response S = Slope of the calibration curve.

3.3.4. Linearity (LOQ to 150%)

To measure linearity, replicate concentration solutions were prepared from main impurity standard concentration, covering a concentration range from about 5% to 150% (0.1, 0.5, 1.0, 2.0, 2.5, and 3.0 ppm). Least‐squares linear regression was applied to a plot of BENZ peak area versus concentration. The linear equation's % y‐intercept was found to be +0.72. Linearity was demonstrated across the concentration range with a correlation coefficient (R) of 0.9996. The regression model (Y = 305.93X + 20462.05) demonstrates homoscedasticity and freedom from systematic bias, as evidenced by a random standardized residual plot with all data points tightly constrained between −0.74 and +1.83. Figure 6B confirmed the method's linearity starting from the LOQ.

3.3.5. Repeatability and Ruggedness

Correctness of the analytical method was evaluated by conducting repeatability and ruggedness studies within and between days. Multiple (for 6 number) doping of BENZ in CTZ samples at limit level of 2 ppm was tested. Repeatability (%RSD both within and between days) and Ruggedness (inter‐analyst and equipment variations) were all within acceptance limits. This proves the method found to be precise and results are tabulated in Table 3A.

3.3.6. Precision at LOQ Level

Precision was assessed by spiking BENZ into solutions at the LOQ concentration level. Table 3B presents the practical relative standard deviation obtained for the spiked BENZ content at the LOQ level. The calculated %RSD for the BENZ content complied with the Q2R2 validation guideline. This demonstrates that the method is precise at the tested LOQ concentration level.

3.3.7. LOD Observation

We analyzed a BENZ solution at the LOD concentration of 0.04 ppm and performed three replicate injections. The outcome of LOD study is shown in Table 3C. Visual detection of the BENZ peak in all three replicates validated the method's LOD as approximately 2%.

3.3.8. Accuracy

The method's accuracy was established by recovery studies, which conformed to the Q2R2 validation guideline. As detailed in Table 3D, test samples (API/Tablet) spiked with BENZ at the LOQ, 100, and 150 percent level concentration of the limit level showed an observed accuracy within the acceptance limit. Based on these results, the analytical procedure is confirmed to be accurate.

3.3.9. Sample Solution Stability

As per ICH requirement, sample solution stability was measured by analyzing the BENZ content in CTZ test and spiked test samples at multiple time intervals. The percentage RSD for BENZ content in the spiked sample was established to be within the accepted limits of analytical validation as detailed in Table 4A.

TABLE 4.

(A) BENZ present at different intervals, (B) Analysis results at variation in method, and (C) Lot evaluation.

(A) BENZ present at different intervals
Analysis time in hours	BENZ present in CTZ in ppm	BENZ present in spiked CTZ in ppm
0	Not present	2.00
18	Not present	1.99
27	Not present	1.99
%RELSTDDEV	Non relevant	0.20
Concentration ranging	Non relevant	Between 1.99 and 2.00
Criteria: ≤ 15%RELSTDDEV

(B) Analysis results at variation in method
Variation	%RELSTDDEV (Replicate = 3)	% RELSTDDEV (Replicates = 9)
Analysis at 40 μL	0.29	0.66
Analysis at 60 μL	0.72	0.66
Mobile phase Flow 0.9 mL	0.54	0.67
Mobile phase Flow 1.1 mL	0.41	0.61
Column oven at 30°	0.45	0.63
Column oven at 40°	0.61	0.66
Mobile phase A OPA 0.05%	0.34	0.69
Mobile phase A OPA 0.15%	0.58	0.67
Criteria: ≤ 15%RELSTDDEV

(C) Lot evaluation
Sample description	Lot used	BENZ present in CTZ in ppm
CTZ API	CTZ Validation Batch‐001	BQL
CTZ API	CTZ Validation Batch‐002	BQL
CTZ API	CTZ Validation Batch‐003	BQL
LCTZ API	LCTZ Validation Batch‐001	BQL
LCTZ API	LCTZ Validation Batch‐002	BQL
LCTZ API	LCTZ Validation Batch‐003	BQL
CTZ Tablet 10 mg (Brand‐Cetzine, Mfd by‐Dr Reddys)	EMV251658	BQL
CTZ Tablet 10 mg (Brand‐Okacet, Mfd by‐Cipla)	5C10937	BQL
CTZ Tablet 10 mg (Brand‐Okacet, Mfd by‐Cipla)	AMO16CPB	BQL
LCTZ Tablet 5 mg (Brand‐Okacet‐L, Mfd by‐Cipla)	AMO311AWA	BQL
LCTZ Tablet 5 mg (Brand‐Levocet, Mfd by‐ Hetrohealthcare)	GT251263A	BQL
LCTZ Tablet 5 mg (Brand‐Lecope, Mfd by‐Mankind Pharma)	D7AG4045	BQL
LCTZ Tablet 10 mg (Brand‐L‐Cetriver 10, Mfd by‐Leeford Pharma)	LCT2011	BQL
LCTZ Tablet 10 mg (Brand‐Levosiz 10, Mfd by‐Systopic Laboratories)	HLT591225	BQL
LCTZ Tablet 10 mg (Brand‐Teczine 10, Mfd by‐Sun Pharma)	SIG1551C	BQL
Criteria: NMT 2 PPM

3.3.10. Robustness

To measure robustness, three different spiked samples were prepared in which BENZ was spiked into CTZ samples at a 100% level (2 ppm) and then analyzed. The percentage RSD for the spiked BENZ content was matched against the method's precision solution, with the results shown in Table 4B, falling within the Q2R2 validation guideline's acceptance limits.

3.3.11. Lot Evaluation

After analyzing three different lots of CTZ and LCTZ drug samples and drug product, no BENZ peaks were found. Therefore, as stated in Table 4C, the final outcome for the batch analysis was reported as Below Quantitation Level (BQL).

4. conclusion

An ultra‐sensitive and reliable HPLC method was successfully established for the quantification of trace levels of BENZ in CTZ and LCTZ drug substance and drug product. Validation results demonstrated acceptable precision, specificity, accuracy, linearity, and robustness, confirming the suitability of the method as per ICH harmonized guideline for Validation of analytical procedures Q2 (R2). Therefore, the method is applicable for release testing of CTZ and LCTZ drug substance and drug product. The novelty of the method is best described in Table 5 below.

TABLE 5.

Comparative summary table of existing and developed method.

Feature	GCHS (Industry standard)	Proposed HPLC method (novel technique)
Matrix effect	Unavoidable (vapor‐phase partitioning)	Avoidable (controlled liquid extraction)
Thermal risk	Moderate (headspace heating)	Negligible (ambient temperature)
Sample handling	Complex (high‐dose feasibility)	Streamlined (high‐ and low‐dose feasibility)
Analyte requirement	Volatility‐dependent	Chromophore‐dependent (UV‐Vis)
Sensitivity	Sensitivity particularly with GC‐MS; however, generic multi‐residual solvent methods may not be optimized for ultra‐trace benzene in specific APIs	Ultra‐sensitive for benzene in cetirizine dihydrochloride and levocetirizine dihydrochloride drug substance and drug product after dedicated optimization, with validated LOD/LOQ suitable for trace‐level quantification below regulatory limits.
Cost	High capital investment for headspace autosampler and GC‐MS instrumentation; higher maintenance and operating costs	Lower instrument and maintenance costs using conventional HPLC systems widely available in pharmaceutical QC laboratories.
Regulatory applicability	Widely accepted by pharmacopeias and regulatory agencies for residual solvent determination according to ICH Q3C and USP < 467 >, limited to drug substance analysis	Fully validated according to ICH Q2 (R2)/Q14 analytical validation principles and suitable as an alternative procedure when equivalent performance is demonstrated for benzene determination in both Cetirizine dihydrochloride and levocetirizine dihydrochloride drug substance and drug product.
Method status	Common technique	Novel and scientifically justified alternative technique

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: HPLC chromatogram showing improper peak elution of BENZ.

Figure S2: HPLC chromatogram of Standard solution showing system suitability parameters.

Figure S3: HPLC chromatogram of Standard, LOD and LOQ solution showing s/n.

Data Availability Statement

Data will be made available on request.