Optimization and Validation of an HPLC Method for the Analysis of Carvedilol and Impurities

Abstract

In this study, a liquid chromatography method was developed and validated for the accurate determination of carvedilol content while minimizing interference from impurity C and N-formyl carvedilol and allowing precise impurity analysis. The reliability of the method was verified through key parameters such as linearity, precision, accuracy, and stability, ensuring robust performance in the detection and quantification of carvedilol and related impurities. And, the method was tested under varying conditions, including changes in the flow rate, initial column temperature, and mobile phase pH. The results showed that the method demonstrated excellent linearity, with R ² values consistently above 0.999 for all analytes. Precision tests yielded RSD% values below 2.0%, confirming the method’s repeatability. Accuracy assessments revealed recovery rates ranging from 96.5% to 101%, while stability studies indicated minimal variation in peak areas and impurity content over extended time periods. These results confirm the method’s reliability for accurate quantification and impurity analysis in pharmaceutical samples.

1. Introduction

Carvedilol, as an adrenergic β-receptor blocker, has blocking effects on both α1 and β receptors and has been widely used in the clinical treatment of cardiovascular diseases such as hypertension, heart failure, and arrhythmias. In recent years, numerous clinical studies on carvedilol have been conducted in various areas, including the prevention of atherosclerosis, protection of cardiac function in patients with different comorbidities, improvement of glycemic control in diabetic patients, prevention and treatment of breast cancer, and anti-Alzheimer’s disease. Furthermore, with the advancement in the research of G protein-coupled receptors (GPCRs), it has been discovered that carvedilol exhibits a unique bias toward β-arrestin-mediated GPCR signaling, which acts selectively on target cells and may reduce the occurrence of adverse drug reactions.

High-performance liquid chromatography (HPLC) is a widely used analytical technique in pharmaceutical and clinical research due to its high resolution, accuracy, and reproducibility. HPLC is particularly well-suited for separating and quantifying compounds in complex mixtures, making it an essential tool in the quality control of pharmaceutical products. Its versatility allows for the detection of a wide range of substances, from small organic molecules to large biomolecules, with excellent sensitivity and specificity. One of the key advantages of HPLC is its ability to provide precise and reliable results, even in the presence of multiple components, impurities, or degradation products. This is especially important in the pharmaceutical industry, where the accurate quantification of active ingredients and impurities is critical for ensuring drug safety and efficacy. HPLC also offers the flexibility of adjusting parameters such as the mobile phase, flow rate, and temperature, enabling the optimization of conditions for specific analytes.

Currently, various methods are available for the detection and quantification of carvedilol, including spectrophotometry and LC-MS/MS. However, these methods often have limitations in terms of sensitivity, selectivity, or the ability to simultaneously quantify carvedilol and its impurities. For instance, spectrophotometric methods may lack the required specificity in the presence of similar compounds and require derivatization steps that can complicate the analysis. The disadvantages of using LC-MS/MS to determine carvedilol include high equipment costs and complex maintenance, which hinder its widespread application in routine laboratories. Although the run time of the LC-MS/MS method is relatively short (∼5 min), its high operational costs (due to instrument maintenance and reagent consumption) and technical requirements for operators remain significant limitations for large-scale routine analysis. The United States Pharmacopoeia USP-NF 2025 contains a method for detecting substances related to carvedilol tablets, with a mobile phase prepared as follows: 1.04 g of sodium dodecyl sulfate was added in 150 mL of buffer (0.7 g of anhydrous monobasic potassium phosphate was dissolved in 500 mL of water and 10 mL of triethylamine was added, with phosphoric acid adjusted to a pH of 3.0 ± 0.1) in a 2-L volumetric flask and sonicated. 720 mL of acetonitrile was added and diluted with water to volume. The mobile phase matching system is relatively complex, using triethylamine and sodium dodecyl sulfate in the preparation process. Triethylamine is extremely volatile and produces pungent ammonia gas, which can cause harm to the human body through the respiratory tract. Sodium dodecyl sulfate is a surfactant and causes significant damage to the chromatographic column. During the detection process, it can reduce the column efficiency, thereby affecting the detection results and increasing the detection cost for enterprises. At the same time, the column temperature reached 40 °C, which can also affect the service life of the chromatographic column. Based on this, the chromatographic method developed in this paper uses conventional phosphate and acetonitrile as the mobile phases. By changing the column temperature of the chromatographic column at different time periods, good impurity separation can be achieved. Such a method not only ensures the consistent quality of carvedilol formulations but also provides a robust tool for pharmacokinetic studies and therapeutic drug monitoring. By enabling precise measurement of carvedilol and its related impurities, a well-optimized HPLC method enhances patient safety and supports the efficacy of carvedilol in various therapeutic contexts.

Moreover, as research continues to explore carvedilol’s broader therapeutic applicationssuch as in atherosclerosis prevention, glycemic control in diabetes, and cancer treatmenta reliable HPLC method becomes even more critical. It facilitates detailed pharmacodynamic studies and helps us to understand the complex interactions of carvedilol with other medications, ultimately contributing to the advancement of clinical practice and research.

2. Experimental Section

2.1. Reagents and Samples

2.1.1. Reagents

Potassium dihydrogen phosphate (AR, Batch No. 20221014) was sourced from the China National Pharmaceutical Group. Phosphoric acid (HPLC grade, Batch No . K1325025) was obtained from Aladdin. Acetonitrile (HPLC grade, Batch No. 23035062) was provided by TEDIA. Sodium hydroxide (AR, Batch No. 20201109), hydrochloric acid (AR, Batch No. 20220516), and 30% hydrogen peroxide (AR, Batch No. 20220325) were all supplied by the China National Pharmaceutical Group.

2.1.2. Samples

Carvedilol tablets (10 mg, Batch No. 2308001KW) and blank excipients for carvedilol tablets (Batch No. 2307001).

2.1.3. Standards

Carvedilol reference standard (99.6%, Batch No. 100730-202303) was sourced from the National Institutes for Food and Drug Control (NIFDC). Impurity C (96.8%, Batch No. 238233C-FZ-01) was obtained from STD. N-Formyl carvedilol (100.0%, Batch No. 1010-RD-0014) was provided by CATO. The structural formulas of the above two compounds are shown in Figure .

1.

Structural formulas of carvedilol (a), impurities C (b), and N-formyl carvedilol (c).

2.2. Instrumentation

The instruments used in the study include a high-performance liquid chromatography (HPLC) system, Agilent 1260, from Agilent; running software: Agilent Control Panel for OpenLab Software 3.6; software: Agilent Data Analysis OpenLab CDS 2.7; an electronic balance, XS205DU, from METTLER TOLEDO; a pH meter, FE28, also from METTLER TOLEDO; and a high-speed centrifuge, LC-LX-H185C, from Shanghai Lichenbangxi.

2.3. Chromatographic Conditions

The HPLC analysis was carried out on an Inertsil ODS-3 V column (4.6 mm ID × 250 mm, 5 μm in particle size). The detection wavelength was set at 240 nm. The sample injection volume was 10 μL, and the flow rate was 1.0 mL/min. Linear gradient elution was performed with 0.02 mol/L potassium dihydrogen phosphate solution (pH adjusted to 2.0 with phosphoric acid) as mobile phase A and acetonitrile as mobile phase B, according to Table . The change of column temperature is also shown in Table .

1. Change of Gradient Elution about the Mobile Phase and Column Temperature.

Time (min)	Mobile Phase A (%)	Mobile Phase B (%)
0	75	25
10	75	25
38	35	65
50	35	65
50.1	75	25
60	75	25
50.1	75	25
60.0	75	25

Column temperature
Time (min)	Temperature (°C)
0	20
20	40
40	20

2.4. Solution Preparation

2.4.1. Preparation of Standard Solution

Carvedilol was prepared by accurately weighing 25 mg in a 50 mL volumetric flask and dissolving it in solvent to make up the volume to 50 mL. 1 mL of this solution was pipetted, and the diluent was added to make exactly 100 mL, with this solution as the standard solution. The impurity C stock solution was prepared by accurately weighing 12.5 mg of the impurity in a 250 mL volumetric flask and dissolving it in ACN to make up the volume to 250 mL. The N-formyl carvedilol stock solution was prepared by accurately weighing 12.5 mg of the impurity in a 25 mL volumetric flask and dissolving it in ACN to make up the volume to 25 mL.

2.4.2. Preparation of Sample Solution

Five tablets of this product were taken into a 100 mL volumetric bottle, an appropriate amount of solvent was added, carvedilol was dissolved by ultrasound, and the solution was diluted with diluent to 100 mL.

2.5. Forced Degradation Studies

In order to determine the selectivity of the unknown degradation product to determine the relevant material law, compulsory degradation research on placebo and samples was carried out under different conditions: acidic (1 N HCl, 1 h, 80 °C), alkaline (1 N NaOH, 1 h, 80 °C), thermic (6 h, 80 °C) and oxidative degradation (3% H₂O₂, 3 h, room temperature), and photolytic (5000 lx + 90 μW, 24 h). The blank samples were prepared without adding the analytes in each stress condition.

2.5.1. Acid Degradation

Five carvedilol tablets were placed in a 100 mL volumetric flask, added with 30 mL of diluent, and sonicated for 15 min with intermittent shaking. After 10 mL of 1 N HCl was added, the mixture was incubated in an 80 °C water bath for 1 h. The solution was neutralized with 10 mL of 1 N NaOH, equilibrated to room temperature, diluted to volume with diluent, mixed, and filtered through a 0.45 μm PVDF membrane prior to analysis.

2.5.2. Alkaline Degradation

Five carvedilol tablets were transferred to a 100 mL volumetric flask, and about 30 mL of diluent was added and sonicated for 15 min with intermittent shaking. A 10 mL portion of 1N NaOH solution was added and kept in an 80 °C water bath for 1 h. Further, for neutralization purposes, 10 mL of 1 N HCl solution was added. The solution was equilibrated at RT, and volume was made up to the mark with diluent, mixed, and filtered through a 0.45 μm PVDF filter for analysis .

Five carvedilol tablets were processed as described in Section , except that 10 mL of 1 N NaOH was added instead of HCl. After 1 h at 80 °C, the solution was neutralized with 10 mL of 1 N HCl, then diluted, mixed, and filtered as above.

2.5.3. Oxidative Degradation

Five carvedilol tablets were processed as described in Section (30 mL of diluent, 15 min of sonication with intermittent shaking). After 10 mL of 3% hydrogen peroxide was added, the mixture was incubated at room temperature for 3 h. The solution was then equilibrated to room temperature, diluted to a volume with diluent, mixed, and filtered through a 0.45 μm PVDF membrane for analysis.

2.5.4. Photolytic Degradation

Five carvedilol tablets were processed as described in Section (30 mL diluent, 15 min sonication with intermittent shaking). The solution was exposed to 4500 ± 500 lx of light for 24 h. After photolysis, it was equilibrated to room temperature, diluted to volume with a diluent, mixed, and filtered through a 0.45 μm PVDF membrane for analysis.

2.5.5. Thermal Degradation

Five carvedilol tablets were processed as described in Section (30 mL diluent, 15 min sonication with intermittent shaking). The mixture was then incubated in an 80 °C water bath for 6 h. After being cooled to room temperature, the solution was diluted to volume with diluent, mixed, and filtered through a 0.45 μm PVDF membrane for analysis.

2.5.6. Nondegraded (CK Group)

Five tablets were placed in a 100 mL volumetric flask, the appropriate diluent was added, and the mixture was sonicated for 15 min with intermittent shaking to dissolve carvedilol. The solution was diluted to volume with diluent, mixed, and filtered, and the filtrate was collected as the nondegraded control.

2.6. Method Validation

2.6.1. System Suitability

Prepare a system suitability solution containing N-formyl carvedilol at approximately 1 μg/mL and impurity C at approximately 0.1 μg/mL for liquid chromatography determination.

2.6.2. Specificity

The operation of forced degradation studies was used.

2.6.3. Limit of Quantification and Limit of Detection

2.6.3.1. Limit of Quantification (LOQ)

The impurity and carvedilol stock solutions were measured accurately, and the mixture was sequentially diluted with solvent to achieve the appropriate concentration. 6 parallel samples were prepared. The prepared LOQ solution concentrations are as follows: carvedilol: 0.0022 μg/mL, impurity C: 0.0035 μg/mL, and N-formyl carvedilol: 0.0076 μg/mL.

2.6.3.2. Limit of Detection (LOD)

3 mL of the LOQ solution was measured accurately, placed in a 10 mL volumetric flask, diluted to volume with solvent, and mixed well.

2.6.4. Linearity and Range

Linearity solutions: the specified volumes of the linearity stock solutions were measured accurately as listed in Table ; these were transfered into the corresponding volumetric flasks, diluted to the mark with solvent, and mixed well, Table .

2. Preparation Method for Linearity Solutions of Different Groups.

Group	Relative concentration (%)	Volume of linearity stock solutions (mL)	Volumetric bottle volume (mL)	Impurity C concentration (μg/mL)	Other substances’ concentration (μg/mL)
L1	25%	1	200	0.025	0.25
L2	50%	1	100	0.050	0.50
L3	100%	1	50	0.100	1.00
L4	150%	3	100	0.150	1.50
L5	200%	2	50	0.200	2.00

2.6.5. Recovery

Accuracy (recovery) was evaluated at four concentration levels: 25%, 50%, 100%, and 150% of the limit concentration. Three replicate test solutions were prepared for each concentration.

2.6.5.1. Blank Solution

5 tablets of the sample were weighed accurately and placed into a 100 mL volumetric flask. A suitable amount of solvent was added, and the mixture was sonicated with intermittent shaking for 15 min to dissolve the carvedilol. The volume was made up with solvent, mixed well, and filtered, and the filtrate was collected.

2.6.5.2. Recovery Solution

5 tablets of the sample were weighed accurately and placed into a 100 mL volumetric flask. A suitable amount of solvent was added, and the mixture was sonicated with intermittent shaking for 15 min to dissolve the carvedilol; 0.5 mL of the impurity mixed stock solution was added, and the volume was made up with solvent, mixed well, and filtered, and the filtrate was collected. The volume of the impurity mixture was changed to 1, 2, and 3 mL to prepare recovery solutions at 50%, 100%, and 150%.

2.6.6. Accuracy

5 tablets of the sample were weighed accurately and placed into a 100 mL volumetric flask. A suitable amount of solvent was added, and the mixture was sonicated with intermittent shaking for 15 min to dissolve the carvedilol; 2 mL of the impurity mixed stock solution was added, and the volume was made up with solvent, mixed well, and filtered, and the filtrate was collected. 6 parallel solutions were prepared.

2.6.7. Robustness

2.6.7.1. Extraction Time

5 tablets of the sample were weighed accurately and placed into separate 100 mL volumetric flasks. A suitable amount of solvent was added to each flask, and the mixture was sonicated with intermittent shaking for 10, 15, and 20 min, respectively, to dissolve the carvedilol. Each solution was diluted to volume with solvent, mixed well, and filtered (material: PVDF, manufacturer: Shanghai Anpu, specifications: 0.45 μm, 25 mm), and the filtrate was collected for analysis.

2.6.7.2. Adsorption of Filter Membrane

100% spiked test solution was taken and centrifuged to obtain the supernatant. Additionally, the solution was filtered using a membrane filter (material: PVDF, manufacturer: Shanghai Anpu, specifications: 0.45 μm, 25 mm). The initial filtrates of 1 mL, 3 mL, and 5 mL were discarded, and the subsequent filtrate was analyzed.

2.6.7.3. Stability of Solution

5 tablets of the sample were weighed accurately and placed into a 100 mL volumetric flask. A suitable amount of solvent was added, and the mixture was sonicated with intermittent shaking for 15 min to dissolve the carvedilol; the volume was made up with solvent, mixed well, and filtered, and the filtrate was collected for analysis.

We investigated the stability of the test solution, control solution (2.4.1), and system suitability solution (2.6.1) at room temperature for 54 h.

2.6.7.4. Variation of Chromatographic Parameters

The effects of chromatographic conditions on the results were investigated by adjusting the flow rate, column temperature, different chromatographic columns, and pH of the mobile phase. The parameters for the variations in the chromatographic conditions are shown in Table .

3. Variations in Chromatographic Conditions.

Parameters	Standard Parameter	Parametric Variation
Flow rate	1.0 mL/min	0.9 mL/min or 1.1 mL/min
Initial column temperature	20 °C	18 or 22 °C
pH of mobile phase	2.0	1.9 or 2.1
Chromatographic column	ODS-3 V 4.6 × 250 mm, 5 μm	Different batches

3. Result

3.1. System Suitability

Figure and Table present the results of the system suitability test. The relative retention times of each chromatographic peak and the resolution between adjacent peaks were calculated according to the reported methods. As shown in Figure , the retention times of carvedilol, impurity C, and N-formyl carvedilol were 22.156, 28.113, and 34.346, respectively. Data in Table show that the RSD% of retention times for all three compounds was less than 2.0% across five consecutive injections. Compared with the control group, the baseline of other injection groups remained stable, with no peak tailing, and the resolution between adjacent peaks was consistent. This indicates that the liquid chromatography method is suitable for systematic analysis in this series of injections, which is further supported by the RRT data..

2.

Liquid chromatograph of system suitability.

4. System Suitability Test Results .

Component	Index	Injection					RSD %
		1	2	3	4	5
Carvedilol	RT (min)	22.194	22.187	22.200	22.180	22.166	0.06
	Peak area	29945.574	29906.463	30064.670	30021.625	29835.607	0.30
	RRT	1.00	1.00	1.00	1.00	1.00	/
	Resolution1	2.71	2.69	2.70	2.71	2.71	/
	Resolution2	6.60	6.62	6.49	6.47	6.45	/
	Tailing factor	1.4	1.4	1.4	1.4	1.4	/
	Theoretical plates	100873	100994	100878	100669	101044	/
Impurity C	RT (min)	28.129	28.119	28.113	28.102	28.081	0.07
	Peak area	3.506	3.689	3.675	3.619	3.590	2.03
	RRT	1.27	1.27	1.27	1.27	1.27	/
	Resolution1	23.76	23.66	23.65	23.57	23.44	/
	Resolution2	1.53	1.52	1.56	1.54	1.51	/
	Tailing factor	1.1	1.1	1.1	1.1	1.1	/
	Theoretical plates	341258	340934	338878	340796	339122	/
N-formyl carvedilol	RT (min)	34.339	34.347	34.342	34.335	34.331	0.02
	Peak area	55.370	55.275	55.252	55.358	55.486	0.17
	RRT	1.55	1.55	1.55	1.55	1.55	/
	Resolution1	10.42	10.38	10.46	10.51	10.45	/
	Resolution2	2.03	2.01	2.03	2.01	2.04	/
	Tailing factor	0.9	0.9	0.9	0.9	0.9	/
	Theoretical plates	67503	67490	66555	66515	66701	/

^aResolution¹ represents the separation from the left peak, and resolution² represents the separation from the right peak, similarly hereinafter.

3.2. Specificity

As shown in Table and Figure , the forced degradation data indicate that excipients in the blank group did not interfere with the main peak or impurity peaks in the sample solution.

5. Results of Strong Degradation Test of Related Substances .

Substance		Group
		CK	Photolytic	Oxidation	Acid	Alkali	Thermal
Main impurity %	Impurity C	-	-	-	-	-	-
	N-formyl carvedilol	0.015	0.017	0.017	0.007	0.063	0.019
Other impurities %	RRT0.21	-	-	-	0.081	-	-
	RRT0.26	-	0.032	-	-	-	-
	RRT0.28	-	-	-	0.179	-	-
	RRT0.71	-	-	-	-	0.025	-
	RRT0.77	-	-	-	-	0.274	0.020
	RRT0.82	-	-	-	-	0.040	-
	RRT0.88	-	-	-	-	0.154	0.021
	RRT0.93	-	-	-	-	0.153	-
	RRT1.05	-	-	-	0.096	-	-
	RRT1.07	-	-	-	-	0.027	-
	RRT1.08	0.019	0.017	0.019	0.070	0.027	0.020
	RRT1.10	-	-	-	-	0.023	0.029
	RRT1.17	-	-	-	-	0.023	-
	RRT1.28	0.035	0.039	0.036	0.037	0.029	0.034
	RRT1.31	-	-	-	-	0.020	-
	RRT1.33	-	-	-	-	0.058	-
	RRT1.37	0.037	0.034	0.036	0.030	0.030	0.035
	RRT1.50	-	-	-	-	0.039	-
	RRT1.60	-	-	-	-	2.579	-
Total impurity %		0.172	0.221	0.186	0.633	3.564	0.280
Total peak area		30036	29630	29942	29959	30050	29642
Material balance %		/	98.6	99.7	99.7	100.0	98.7
Main peak purity		999.389	999.410	999.447	999.375	999.450	999.374
Minimum separation degree		6.78	6.40	6.80	2.95	5.84	2.87

^aMaterial balance % is calculated as the sum of the percentage of intact parent drug (carvedilol) and its degradation products relative to the initial concentration (100%) before forced degradation. It reflects the completeness of analyte recovery during degradation, ensuring no significant loss of drug-related substances due to volatilization, adsorption, or irreversible degradation, thereby validating the reliability of degradation product profiling.

3.

Liquid chromatograph of specificity.

Under all conditions, the separation between impurities and the main peak was greater than 1.5 (USP resolution). The minimum resolution observed in the acid degradation group was 2.95. The purity of the main peak was consistently above 999.000. These results demonstrate that the method meets the acceptance criteria.

3.3. LOD and LOQ

The values of LOQ and LOD were calculated using the Agilent Data Analysis OpenLab CDS 2.7 software that comes with the liquid phase. Figure and Table present the results of LOD and LOQ. The LOD and LOQ concentrations of this method comply with conventional analytical standards. The S/N for LOD exceeds 3, and for LOQ, it is above 10. This indicates that the method can effectively detect and accurately quantify carvedilol, impurity C, and N-formyl carvedilol at low concentrations. The concentration range is sufficient to meet sensitivity requirements, ensuring proper separation and accurate quantification of the impurities and the main component. Overall, the method is reliable and can stably and accurately detect and quantify target substances in complex samples.

4.

Liquid chromatographs of LOD and LOQ.

6. Results of LOD and LOQ of Related Substances .

Indicator	Index	Substance
		Carvedilol	Impurity C	N-formyl carvedilol
LOD	Peak area	0.378	0.159	0.443
	S/N	10.5	3.8	4.6
	Concentration (μg/mL)	0.0022	0.0035	0.0076
	Equivalent to sample concentration%	0.0004	0.0007	0.0015
LOQ	Peak area	0.551	0.585	1.535
	S/N	11.2	10.1	11.4
	Concentration (μg/mL)	0.0075	0.0117	0.0253

^aS/N values for LOD (≥3) and LOQ (≥10) meet ICH Q2 (R1) guidelines. The differences in S/N across analytes are attributed to their distinct UV absorbance properties (e.g., molar absorptivity), but all exceed the required thresholds to ensure reliable trace detection.

The results in Table indicate that the repeatability of the LOQ for carvedilol, impurity C, and N-formyl carvedilol is satisfactory, with average peak areas of 0.546, 0.574, and 1.509, respectively. The RSD% values are 3.34% for carvedilol, 2.99% for impurity C, and 1.11% for N-formyl carvedilol, demonstrating low variability across the six repetitions. Overall, these results confirm that the method provides consistent and reliable quantification at the LOQ level.

7. Results of the Quantitative Limit Repeatability Investigation of the Relevant Substances.

Group	Carvedilol		Impurity C		N-formyl carvedilol
	Peak area	S/N	Peak area	S/N	Peak area	S/N
LOQ-1	0.551	11.2	0.585	10.1	1.535	11.4
LOQ-2	0.549	18.6	0.554	16.3	1.491	18.8
LOQ-3	0.516	17.7	0.563	16.4	1.511	19.0
LOQ-4	0.568	13.9	0.559	12.5	1.522	14.4
LOQ-5	0.536	12.4	0.587	11.7	1.499	13.4
LOQ-6	0.558	12.6	0.595	11.3	1.498	13.1
Average	0.546	/	0.574	/	1.509	/
RSD%	3.34	/	2.99	/	1.11	/

3.4. Linearity

Figure displays the chromatograms of carvedilol, impurity C, and N-formyl carvedilol at different concentration levels. Figure shows that the measurement results for the three substances exhibit a linear relationship within a limited range. By plotting the calibration curves of the analyte concentration versus the peak area, the slope, intercept, and correlation coefficient were calculated. The results indicate that in the first set of fitting equations, the R ² values for all three substances are no less than 0.999. In the second set of fitting equations, the R ² values for all three substances are equal to 1. This demonstrates that all results meet the detection standards of the Chinese Pharmacopoeia (ChP). Meanwhile, by dividing the linear slope of carvedilol by the linear slopes of impurity C and N-formyl carvedilol, the correction factors for the two impurities are obtained as 1.2 and 1.1, respectively.

5.

Liquid chromatograph of linearity.

6.

Linear equations for impurity C, carvedilol, and N-formyl carvedilol.

3.5. Accuracy

The results from Figure , Tables and indicate that the recovery rates for impurity C and N-formyl carvedilol show good recovery performance at different concentrations. For impurity C, the average recovery rate ranges from 95.7% to 99.3%, with RSD% values below 2, demonstrating good repeatability. The recovery rates for N-formyl carvedilol range from 99.9% to 101.5%, with an average recovery rate of 100.6% and an RSD% as low as 0.84, indicating high precision. Overall, these results demonstrate that the method employed is efficient and reliable for detecting both substances and meets the relevant standard requirements. The stability of the recovery rates further enhances the credibility of this analytical method.

7.

Liquid chromatograph of accuracy.

8. Results of the Recovery Rates of Impurity C .

Concentration	Group	Content1 (μg/mL)	Content2 (μg/mL)	Recovery %	Average %	RSD %
25%	1	0.0234	0.0224	95.57	96.5	1.79
	2		0.0230	98.24
	3		0.0224	95.57
50%	1	0.0469	0.0448	95.62	95.7
	2		0.0449	95.79
	3		0.0448	95.62
100%	1	0.0937	0.0902	96.26	97.7
	2		0.0921	98.24
	3		0.0925	98.71
150%	1	0.1406	0.1404	99.91	99.3
	2		0.1405	99.97
	3		0.1378	98.04

^aContent¹ represents the added value, and content² represents the measured value.

9. Results of Recoveries of N-Formyl Carvedilol .

Concentration	Group	Content1 (μg/mL)	Content2 (μg/mL)	Recovery %	Average %	RSD %
25%	1	0.2534	0.3279	100.60	101.0	0.84
	2		0.3271	100.29
	3		0.3316	102.07
50%	1	0.5068	0.5872	101.45	101.5
	2		0.5852	101.06
	3		0.5893	101.88
100%	1	1.0136	1.0863	99.97	99.9
	2		1.0877	100.12
	3		1.0812	99.47
150%	1	1.5204	1.5985	100.34	100.0
	2		1.5918	99.90
	3		1.5921	99.91

^aContent¹ represents the added amount, and content² represents the measured amount.

3.6. Repeatability

The results in Tables and demonstrate the repeatability levels for impurity C, N-formyl carvedilol, and other impurities. In the repeatability tests, the value for impurity C remained consistent, with an average of 0.018% and no variability. The average percentage for N-formyl carvedilol was 0.218%, with an RSD% of 0.860, indicating good precision. The average for other impurities was 0.037%, which remained stable throughout the tests. The total impurity average was 0.389%, with an RSD% of 1.480, suggesting that the method performs well in terms of repeatability.

10. Repeatability Test Results for Related Substances.

Group	Impurity C %	N-formyl carvedilol %	Other impurities %	Total impurity %
1	0.018	0.218	0.037	0.390
2	0.018	0.217	0.037	0.387
3	0.018	0.216	0.037	0.384
4	0.018	0.216	0.036	0.383
5	0.019	0.221	0.037	0.399
6	0.018	0.217	0.037	0.388
Average	0.018	0.218	0.037	0.389
RSD %	/	0.860	/	1.480
Tmsv-Dif	0.001	/	0.001	/

11. Intermediate Precision Test Results of Related Substances.

Group	Impurity C %	N-formyl carvedilol %	Other impurities %	Total impurity %
1	0.018	0.214	0.036	0.372
2	0.019	0.215	0.035	0.375
3	0.018	0.215	0.035	0.373
4	0.018	0.214	0.035	0.374
5	0.018	0.216	0.036	0.379
6	0.018	0.215	0.035	0.377
Average	0.018	0.215	0.035	0.375
RSD %	/	0.350	/	0.70
Tmsv-Dif	0.001	/	0.001	/

In the intermediate precision tests, the average for impurity C remained at 0.018%, showing good stability. The average percentage for N-formyl carvedilol was 0.215%, with an RSD% of 0.350, indicating high intermediate precision. The average for the other impurities was 0.035%, demonstrating good stability. The total impurity average was 0.375%, with an RSD% of 0.70, further confirming the reliability of the method. Overall, this analytical method exhibits excellent performance in terms of both repeatability and intermediate precision.

3.7. Durability

3.7.1. Extraction Duration

The results in Table indicate that different extraction times have a minimal impact on the measurement results for impurity C, N-formyl carvedilol, and other impurities. As the extraction time increases, the content of N-formyl carvedilol shows a slight increase, but the percentages of other impurities and total impurities remain relatively stable. Overall, the extraction times between 10 and 20 min yield consistent measurement results for impurities, suggesting that the extraction efficiency within this time range is uniform.

12. Effect of Different Extraction Times on the Test Results of the Samples.

Group	Impurity C %	N-formyl carvedilol %	Other impurities %	Total impurity %	Impurity amount count
10 min	-	0.014	0.036	0.150	13
15 min	-	0.015	0.036	0.151	13
20 min	-	0.015	0.036	0.154	13

3.7.2. Adsorption of Filter Membrane

The results in Table indicate that the adsorption effect of the filter membrane on the sample test results is minimal. The content of impurity C remains between 0.018% and 0.019% across different sample volumes, with a maximum difference value of 0.001. The content of N-formyl carvedilol ranges from 0.216% to 0.218%, with a maximum difference value of 0.002. The total impurity content varies between 0.368% and 0.381%, indicating that the adsorption effect of the filter membrane has a limited impact on the test results.

13. Effect of Filter Membrane Adsorption on Sample Test Results.

Group	Index	Centrifugation	1 mL	3 mL	5 mL
Impurity C %	Content	0.018	0.017	0.018	0.019
	Difference value	/	0.001	0.000	0.001
N-formyl carvedilol %	Content	0.218	0.216	0.217	0.218
	Difference value	/	0.002	0.001	0.000
Other impurities %	Content	0.037	0.034	0.036	0.036
	Difference value	/	0.003	0.001	0.001
Total impurity %	Content	0.381	0.368	0.372	0.378
	Difference value	/	0.013	0.009	0.003

^aWith the centrifugation group, centrifugal speed is 6000 rpm/min.

3.7.3. Stability of Solution

Based on the results from Tables , , and , the samples demonstrate good stability at different time points. Table shows that the peak area of impurity C remains around 4.660 over 54 h, with an RSD of 0.44%, indicating minimal variation. Table ‘s system suitability test reveals that the peak area of carvedilol is stable at 27,758.607, while the separation of N-formyl carvedilol consistently remains at 7.28. Table indicates that the total impurity percentage fluctuates between 0.151% and 0.168%, and the content of N-formyl carvedilol stays between 0.015% and 0.036%. In summary, the method shows good reliability and consistency in sample stability and impurity detection, making it suitable for practical analysis.

14. Test Results for the Stability of the Standard Solution.

Group	Impurity C		N-formyl carvedilol		Carvedilol
	Peak area	RSD %	Peak area	RSD %	Peak area	RSD %
0 h	4.662	0.44	60.352	0.12	64.735	0.22
10 h	4.706		60.416		64.713
25 h	4.694		60.441		65.059
36 h	4.655		60.438		64.813
47 h	4.677		60.380		64.972
54 h	4.660		60.568		64.978

15. Results of Tests for the Stability of Substance Solution-System Suitability .

Group	Component	Peak area	Ratio to CK %	Separation1	Separation2
0 h (CK)	Carvedilol	27669.904	/	2.55	6.06
	Impurity C	4.599	/	21.46	1.37
	N-formyl carvedilol	62.338	/	7.26	1.98
10 h	Carvedilol	27758.607	100.3	2.52	6.12
	Impurity C	4.602	100.1	21.29	1.37
	N-formyl carvedilol	62.470	100.2	7.28	1.96
25 h	Carvedilol	27731.836	100.2	2.42	6.11
	Impurity C	4.581	99.6	21.40	1.35
	N-formyl carvedilol	62.641	100.5	7.24	1.94
36 h	Carvedilol	27677.742	100.0	2.46	6.07
	Impurity C	4.603	100.1	21.09	1.36
	N-formyl carvedilol	62.510	100.3	7.26	1.96
47 h	Carvedilol	27664.006	100.0	2.43	6.08
	Impurity C	4.607	100.2	21.16	1.35
	N-formyl carvedilol	62.853	100.8	7.12	1.96
54 h	Carvedilol	27625.598	99.8	2.46	6.07
	Impurity C	4.627	100.6	21.73	1.40
	N-formyl carvedilol	62.438	100.2	7.38	1.98

^aSeparation¹ and separation² are as defined in Table . Ratio to CK %: percentage of analyte content in degraded samples relative to the nondegraded control group (CK), where CK is set as 100%.

16. Results of Solution Stability Test of Test Products.

Group	Impurity C %	N-formyl carvedilol %	Other impurities %	Total impurity %
0 h	-	0.015	0.036	0.151
9 h	-	0.015	0.035	0.154
25 h	-	0.014	0.035	0.167
36 h	-	0.015	0.036	0.168
47 h	-	0.015	0.036	0.168
54 h	-	0.015	0.036	0.168

3.7.4. Chromatographic Parameter Adjustment

As shown in Figure , the RT of the three tested solutions exhibited only slight shifts under minor changes in the flow rate, initial column temperature, and the pH of the mobile phase. However, the peak shape and separation were still well-maintained.

8.

Liquid chromatograms of method durability: (A) system applicability group, (B) standard group, and (C) sample group to be tested.

The results from Tables and demonstrate that this method shows high stability for the detection of the three major substances: carvedilol, impurity C, and N-formyl carvedilol. Regardless of variations in the flow rate, initial column temperature, or mobile phase pH, the peak areas and separation factors of the three substances showed minimal changes, indicating the excellent robustness of the method under different conditions. Particularly for N-formyl carvedilol, the variation in the peak area remained within 1%, strongly supporting the reliability of the method.

17. Statistics of Solution Results Related to Substance Durability–System Suitability .

Chromatographic parameter		Component	RT, min	RRT	Peak area	Separation1	Separation2
Standard Parameter		Carvedilol	22.086	1.00	30001.635	2.80	6.51
		Impurity C	28.052	1.27	4.679	24.02	1.63
		N-formyl carvedilol	34.354	1.56	61.498	6.46	/
Flow rate mL/min	0.9	Carvedilol	22.873	1.00	32033.033	2.62	6.30
		Impurity C	28.750	1.26	5.526	23.60	1.46
		N-formyl carvedilol	35.355	1.55	68.828	9.44	2.15
	1.1	Carvedilol	21.399	1.00	27783.742	2.90	7.44
		Impurity C	27.428	1.28	4.328	24.05	1.85
		N-formyl carvedilol	33.495	1.57	56.219	9.58	1.95
Initial column temperature °C	18	Carvedilol	22.170	1.00	29850.033	2.51	6.45
		Impurity C	27.982	1.26	4.719	23.07	1.49
		N-formyl carvedilol	34.410	1.55	61.872	7.05	1.81
	22	Carvedilol	21.944	1.00	30101.775	2.76	7.34
		Impurity C	28.002	1.28	4.626	23.98	1.69
		N-formyl carvedilol	34.261	1.56	61.320	11.01	2.17
pH of mobile phase	1.9	Carvedilol	22.142	1.00	30016.613	2.80	6.51
		Impurity C	28.051	1.27	4.886	23.62	1.80
		N-formyl carvedilol	34.390	1.55	62.089	8.06	2.02
	2.1	Carvedilol	21.866	1.00	30244.555	2.70	7.28
		Impurity C	27.892	1.28	4.644	24.56	1.56
		N-formyl carvedilol	34.255	1.57	62.478	8.46	2.04
Column number #42		Carvedilol	22.007	1.00	27741.805	2.55	6.07
		Impurity C	27.976	1.27	4.642	21.49	1.41
		N-formyl carvedilol	34.224	1.56	62.684	5.06	1.96

^aSeparation¹ and separation² are as defined in Table .

18. Durability of the Substance in QuestionResult of Impurity Content in Test Solution.

Chromatographic parameter		Content or difference	Impurity C	N-formyl carvedilol	Other impurities	Unknown total impurity	Total impurity
Standard Parameter		Content %	0.018	0.218	0.037	0.154	0.391
Flow rate mL/min	0.9	Content %	0.018	0.218	0.036	0.157	0.393
		Difference %	0.000	0.000	0.001	0.003	0.002
	1.1	Content %	0.018	0.217	0.037	0.142	0.377
		Difference %	0.000	0.001	0.000	0.012	0.014
Initial column temperature °C	18	Content %	0.018	0.217	0.037	0.153	0.388
		Difference %	0.000	0.001	0.000	0.001	0.003
	22	Content %	0.019	0.217	0.037	0.153	0.389
		Difference %	0.001	0.001	0.000	0.001	0.002
pH of mobile phase	1.9	Content %	0.018	0.217	0.038	0.150	0.385
		Difference %	0.000	0.001	0.001	0.004	0.006
	2.1	Content %	0.018	0.216	0.036	0.146	0.381
		Difference %	0.000	0.002	0.001	0.008	0.010
Column number #42		Content %	0.018	0.214	0.036	0.146	0.372
		Difference %	0.000	0.004	0.001	0.008	0.019

^aDifference represents the absolute value of the difference between this group and the value measured under standard chromatographic conditions.

Although there were slight fluctuations in the detection of total impurities with changes in flow rate and pH, the maximum difference was only 0.014%, which is within acceptable limits. This demonstrates that the method not only provides accurate detection of major components but also delivers stable and reliable results in impurity analysis, proving its strong applicability and robustness.

4. Discussion

The liquid chromatography method developed for the analysis of carvedilol, impurity C, and N-formyl carvedilol shows clear advantages when compared to traditional methods for carvedilol determination. Conventional techniques, such as UV spectrophotometry or older chromatographic methods, often lack the specificity and sensitivity required for comprehensive impurity profiling. This problem usually needs to be solved by derivation reactions. In contrast, the method described in this study not only provides precise quantification of the main component but also allows for the reliable detection of trace impurities, which is critical in pharmaceutical quality control.

Previous methodologies were more susceptible to fluctuations in experimental parameters like flow rate or mobile phase composition. The robustness of the current method was validated by intentionally varying conditions, such as flow rate, initial column temperature, and mobile phase pH, without significant changes in peak area or retention time for the three analytes. This level of stability is especially advantageous for ensuring consistency across different analytical setups, a limitation in earlier methods.

The sensitivity of the method also marks a significant enhancement over that of older techniques. Conventional methods typically lack the detection limits needed to accurately quantify low-level impurities, often resulting in incomplete impurity profiles. The LOD and LOQ values achieved in this study are well within the acceptable ranges for pharmaceutical analysis, ensuring that even trace amounts of impurity C and N-formyl carvedilol can be reliably detected and quantified. This improvement is particularly important for meeting regulatory guidelines, which have become increasingly stringent with regard to impurity levels in drug substances.

In addition, the accuracy and recovery rates have been significantly improved. Traditional methods often suffered from lower recovery rates, particularly at different concentration levels. The current method demonstrates high recovery for impurity C and N-formyl carvedilol, with recoveries consistently ranging between 96.5% and 101%. This precision in recovery ensures that the method can be reliably used for routine quality control as well as in research environments, where accurate quantification is essential.

Furthermore, the method offers enhanced repeatability and intermediate precision compared to older techniques. The RSD values across multiple injections and testing conditions remain low, indicating that the method provides consistent results over time. This is particularly valuable in the context of pharmaceutical manufacturing, where batch-to-batch consistency is critical for ensuring product quality and regulatory compliance.

In conclusion, the liquid chromatography method developed for carvedilol, impurity C, and N-formyl carvedilol represents a significant improvement over traditional methods. Its robustness, sensitivity, and accuracy make it a superior option for pharmaceutical analysis, providing reliable and consistent results, even under varying conditions. These advancements highlight the method’s potential for broader application in the pharmaceutical industry, ensuring adherence to modern regulatory standards and improving the overall quality control process.

5. Conclusion

The liquid chromatography method developed in this study provides a significantly better analytical approach than traditional methods for the determination of carvedilol and its impurities, achieving important improvements in robustness, sensitivity, and accuracy. As a key drug for treating cardiovascular diseases such as hypertension, heart failure, and angina, the safety and efficacy of carvedilol in clinical applications highly depend on precise monitoring of trace impurities in the drug. Through system testing, it has been confirmed that this method performs stably under different experimental conditions and can accurately detect trace impurities, which is crucial for ensuring the quality of carvedilol preparations and patient medication safety.

The reliable method developed in this study will directly affect the quality control practices of the pharmaceutical industry: high sensitivity and accuracy enable pharmaceutical companies to more effectively monitor the impurity spectrum in the production process of carvedilol, ensuring that products continue to comply with increasingly stringent global drug regulatory standards (such as ICH Q3D impurity control guidelines) and reducing compliance risks and product recall possibilities caused by excessive impurities.

In the drug development and manufacturing stages, the precise impurity data provided by this method help to gain a deeper understanding of the production process and identify and control the sources of impurities, thereby optimizing the production process and improving product quality and yield.

Meanwhile, its excellent robustness and repeatability make this method an ideal tool for pharmaceutical companies to establish robust and reliable quality control systems, providing strong support for the full chain quality monitoring of carvedilol and its formulations from raw materials to finished products.

This method not only achieves breakthroughs in analytical performance at the technical level but also becomes a highly valuable tool in the research and manufacturing environment due to its strong ability to ensure the quality of the key cardiovascular drug carvedilol as well as its significant contribution to improving quality control efficiency, meeting regulatory requirements, and ensuring drug safety in the pharmaceutical industry. Ultimately, it serves the goal of enhancing the patient medication safety and public health.

Data are provided within the manuscript files.

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L.Y. and W.L. contributed to this work and should be regarded as co-first authors. H.Z. and Y.Y. contributed to this work equally and should be regarded as co-corresponding authors.

The authors declare no competing financial interest.