Determination of Rhodamine B in Chili Powder by WAYEAL LCMS-TQ9200 LCMS/MS System

Rhodamine B (also referred to as Rose Red B or Basic Violet 10) is a synthetic basic fluorescent dye that belongs to the xanthene chemical class. This substance itself exhibits a bright rose-red color and is readily soluble in polar solvents such as water and ethanol. Initially, it was widely used in industrial fields including textiles, printing, and fluorescent labeling. Due to its low cost and strong coloring ability, some unscrupulous merchants have illegally added it to food products for dyeing purposes, such as chili powder, Sichuan peppercorns, meat products, and seasonings, making it an important contaminant that endangers food safety.
Rhodamine B is clearly toxic to the human body. It can enter the body via the digestive tract or skin, interfering with normal physiological metabolism. Additionally, it poses potential carcinogenic and mutagenic risks. As a result, it has been classified as a non-edible substance prohibited in food by multiple countries. It is a key target of food safety supervision, and its detection rate remains persistently high in various seasonings and processed foods.

In this application note, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of Rhodamine B in food was established using the WAYEAL LCMS-TQ9200 liquid chromatography-tandem mass spectrometry system. This method achieves efficient extraction, precise separation, and sensitive quantification of the target analyte while effectively eliminating food matrix interferences. It offers reliable technical support for rapid screening and accurate quantification of Rhodamine B in food, thereby facilitating effective food safety supervision.

Keywords: Food safety, Rhodamine B, LCMS/MS

1. Instruments and Reagents

1.1 Instrument Configuration

Table 1 Instrument Configuration List

1.2 Reagents and Standards List

Table 2 Reagents and Standard List

1.3 Experiment Material and Auxiliary Equipment

Vortex mixer

High-speed centrifuge

Analytical balance

Solid phase extraction (SPE) manifold (with vacuum pump)

Nitrogen evaporator

Orbital shaker

2. Experiment Method

2.1 Solution Preparation

2.1.1 0.1% Formic Acid in Water: Transfer 1mL of formic acid into 500mL of water, mix thoroughly, and bring to a final volume of 1000 mL with water.

2.1.2 0.1% Formic Acid in Acetonitrile: Transfer 1mL of formic acid into 500mL of acetonitrile, mix thoroughly, and fix to the volume of 1000mL with acetonitrile.

2.1.3 50% Methanol in Water: Transfer 500mL of methanol accurately into a 1L volumetric flask, then fix to volume with water.

2.1.4 50% Methanol in Water with 0.1% Formic Acid: Transfer 1mL of formic acid into 500mL of 50% methanol in water, mix thoroughly, and dilute to the volume of 1000 mL with 50% methanol in water.

2.1.5 5% Ammonia in Methanol: Transfer 5mL of ammonia solution into a 100mL volumetric flask, dilute to volume with methanol, and mix thoroughly.

2.2 Sample Pretreatment

2.2.1 Sample Extraction

Accurately weigh 1g of chili powder sample into a 50mL plastic centrifuge tube. Accurately add 10.0mL of 50% methanol aqueous solution containing 0.1% formic acid, and mix well. Add two ceramic homogenizers, then vortex for 15 min on a vortex mixer. Centrifuge at 8000 r/min for 10min. Transfer 3mL of the supernatant (extract layer) and filter through a 0.45μm organic-phase filter membrane before purification.

2.2.2 Sample Purification

NovaPre MCX Solid Phase Extraction (SPE) Cartridge Conditioning: Before use, sequentially activate the cartridge with 3mL of methanol and 3mL of water.

Accurately transfer 1.0mL of the extract onto the NovaPre MCX solid phase extraction (SPE) cartridge. Sequentially wash the cartridge with 3mL of 0.1% formic acid in water, 3 mL of water, and 3mL of methanol. Elute the target analyte with 6mL of ammonia in methanol solution, and collect the eluate. Evaporate the eluate to near dryness under a stream of nitrogen at 45°C. Dissolve the residue in 0.5mL of the initial mobile phase, filter through a 0.22μm organic-phase filter membrane, and then inject the filtrate for instrumental analysis.

2.3 Experiment Conditions

2.3.1 HPLC Conditions

Chromatographic column: C18, 1.7μm, 2.1*50mm;
Mobile phase: Phase A: 0.1% formic acid in water; Phase B: 0.1% formic acid in acetonitrile;
Flow rate: 0.4 mL/min;
Column temperature: 40 °C;
Injection volume: 1µL.

2.3.2 Mass Spectrometry Conditions

Table 3 Compound Mass Spectrometry Parameters

Note: * Quantifier ion.

3. Experiment Result

3.1 Standards Chromatogram

The detection of Rhodamine B was completed within 7 minutes. As shown in Figure 1, the compound peak exhibited good peak shape and satisfactory response, meeting the requirements for experimental determination.

Fig 1 Chromatogram of Rhodamine B

3.2 Linear Range

An appropriate amount of Rhodamine B standard working solution was accurately transferred and diluted with the initial mobile phase to prepare the standard curve. The linear range was 0.1–10ng/mL. The deviation between the linear results and the known concentrations was within the maximum allowable deviation. The coefficient of determination (R²) was greater than 0.999, indicating good linearity.

Fig 2 Rhodamine B Standard Curve

3.3 Limit of Quantification and Limit of Detection

In this method, the limit of detection (LOD) and limit of quantification (LOQ) for Rhodamine B were established at 0.1 ng/mL and 0.5 ng/mL, respectively. The corresponding signal-to-noise (S/N) ratios are summarized in the table below. The S/N ratios at the LOD exceeded 3, while those at the LOQ exceeded 10.

Table 4 Limit of Quantification and Limit of Detection

Fig 3 Signal-to-Noise Ratio at LOD for Rhodamine B (0.1ng/mL)

Fig 4 Signal-to-Noise Ratio at LOQ for Rhodamine B (0.5ng/mL)

3.4 Repeatability

Standard solutions at three concentrations (0.5, 2, and 5ng/mL) were injected six times consecutively. The results are shown in the table below. The relative standard deviations (RSDs) of the retention time and peak area for Rhodamine B at low, medium, and high concentrations were all within 5%, meeting the experimental requirements.

Table 5. Repeatability Test for Rhodamine B at Low, Medium, and High Concentrations

Fig 5 Chromatograms of 6 Consecutive Injections of 0.5ng/mL Rhodamine B

Fig 6 Chromatograms of 6 consecutive injections of 2ng/mL Rhodamine B

Fig 7 Chromatograms of 6 consecutive injections of 5ng/mL Rhodamine B

3.5 Spike Recovery

Sample A was spiked with a standard solution to increase the Rhodamine B concentration by 2ng/mL. Six replicate injections were then analyzed. The obtained recovery was 105.7% with an RSD of 1.473%, satisfying the method acceptance criteria.

3.6 Carryover

After injecting the 10ng/mL standard solution, a blank solvent was injected to evaluate carryover. The results are shown in the figure below. The carryover in the blank was less than 10% of the lowest calibration standard (0.1ng/mL), meeting the experimental requirements.

Fig 8 Blank Chromatogram of the Compound

3.7 Sample Test

The Rhodamine B concentration in Sample A was below the method detection limit (MDL).

Fig 9 Chromatogram of Rhodamine B in Sample A

4. Conclusion

In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of Rhodamine B was established using the WAYEAL LCMS-TQ9200 system. The obtained data demonstrated that the method produced chromatographic peaks with good symmetry and no tailing, and the sensitivity met the experimental requirements. The coefficient of determination (R²) was greater than 0.999, satisfying the linearity criteria. The repeatability (RSD) at low, medium, and high concentrations was within 5%, and the carryover in the blank was less than 10% of the lowest calibration standard. These results indicate that the method, when coupled with the WAYEAL LC-MS/MS system, is capable of meeting the routine qualitative and quantitative detection requirements for target analytes.