A QTRAP-based mass spectrometry method for the detection and confirmation of nine sweeteners in Chinese rice wine

A rapid and sensitive QTRAP mass spectrometric method was developed for the detection and confirmation of nine sweeteners (acesulfame, saccharin sodium, sodium cyclamate, aspartame, alitame, sucralose, neotame, stevia extract, and advantage) in Chinese rice wine. The samples with different sugar contents were diluted with ultrapure water, and the standard working solution was prepared by adding 1.0 mL of Chinese rice wine blank matrix to the standard series solution. The target was separated by a Kinetex 2.6 μm F5 100 A LC Column 100 × 2.1 mm chromatographic column, MRM-IDA-EPA by applying QTRAP mass spectrometry composite mode acquisition was applied to obtain MRM data as well as highly sensitive secondary fragmentation full-spectrum data at the same time to establish a standard library of nine Chinese rice wine sweeteners for accurate characterization and MRM data peak area (external standard method) quantification by automatic comparison with the library of standards. The experiments showed that the correlation coefficients of the standard curves of the nine sweeteners were greater than 0.997 in the range of 10 ~ 500 µg/L standard working series solutions, and the limits of quantification of the method were in the range of 0.8–88.0 µg/kg, with spiked recoveries of 80.3%~118.8% and relative standard deviations (RSDs) of 0.74%~6.40%. QTRAP mass spectrometry was used for the determination of the nine sweeteners in Chinese rice wine with easy and fast pretreatment, simple operation, high accuracy, and sensitivity.


Introduction
Sweeteners are commonly used additives in the food industry [1], and in a large counterfeit "Shaoxing Chinese rice wine" case uncovered by Shaoxing police at the end of 2019, the counterfeit Chinese rice wine (CRW) was mainly made of raw wine with added flavoring and sweeteners, detection methods is meaningful.Long-term consumption of sweeteners may lead to metabolic system diseases such as hyperglycemia, glucose intolerance, obesity, and nervous system abnormalities such as irritability, depression, memory decline, Alzheimer's disease, and even genetic toxicity and carcinogenicity.In recent years, the counterfeiting of rice wine has been repeatedly prohibited.Therefore, risk monitoring of sweeteners in rice wine is of great significance to promote the development of local industries, prevent and control food safety hazards, and ensure the health of the people.Therefore, risk monitoring of sweeteners in CRW is important to promote the development of local industries, prevent and control food safety hazards, and protect the health of the people.
The determination of sweeteners in CRW by the abovementioned standards has two defects: first, there are too many standards with different pretreatment methods involving instruments such as gas chromatography [15], liquid chromatography [16], and liquid chromatography-triple quadrupole tandem mass spectrometry, which wastes considerable human, material and financial resources; second, sucralose in CRW is determined by evaporative light scattering in the abovementioned method [16], and the evaporative light scattering detector is a general-purpose detector that can detect any sample with lower volatility than the mobile phase without the need for the sample to contain chromogenic groups.The evaporative light scattering detector is more sensitive than the differential refractive index detector, insensitive to temperature changes, has a stable baseline, and is suitable for use in combination with gradient elution liquid chromatography.However, the matrix of CRW is complex, and its rich carbohydrates (oligosaccharides) and amino acids all interfere with the detection of sucralose and are prone to false positives.during the determination of sucralose in CRW by liquid chromatographytandem mass spectrometry, the detection of sweeteners at low concentrations faces qualitative difficulties.In MRM mode, although the quantitative ion pair 178 > 79.9 (ESI -) can meet the quantitative limit of quantification, the qualitative ion pair 202 > 122 (ESI + ) has a weak response and cannot be accurately characterized.The existing reports [17] on the detection of sweeteners in CRW by high-performance liquid chromatography-tandem mass spectrometry mostly use volatile alcohol followed by dilution injection, and there are also direct dilution injections, but because CRW is rich in amino acids, vitamins, oligosaccharides, polyphenols, and minerals, matrix effects are inevitable, which affect the sensitivity, recovery, and accuracy of characterization.The development of a rapid and sensitive method for the analysis of multiple sweeteners is particularly important to ensure the safety of CRW consumption.In LC-MS/MS, the matrix effect is due to the competition for a charge between nontarget analytes flowing out of the spray needle together with the target, which attracts the generated droplets firmly together and prevents them from splitting into smaller microdroplets, changing the surface tension of the charged droplets, thus leading to a decrease or enhancement of the ionization efficiency of the target, causing a decrease or increase in the response, which gives rise to the so-called matrix inhibition effect or matrix enhancement effect [18].Quadrupole ion trap (QTRAP) mass spectrometry is becoming the first choice for the detection of trace or trace components due to its high sensitivity and strong anti-interference capability.QTRAP mass spectrometry is more sensitive than high-performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) in sweetener detection.Koyama [19] detected ACS-K, SAC, CYC, ASP, and STV by QTRAP mass spectrometry with an LOQ of 1 mg/kg.The LOQs of SCL, DUL, GA, and REB were 5 mg/kg.For confirmatory analyses in food safety regulations, QTRAP mass spectrometry can provide qualitative and quantitative information through the multiresponse monitoring (MRM) mode to improve the sensitivity and selectivity of the analysis.Therefore, liquid chromatography/tandem mass spectrometry (LC-MS/MS) has become a common tool for the detection of various sweeteners in food and environmental water in recent years [20].Scheurer [21] developed an LC-MS/MS method to analyze seven sweeteners in wastewater and surface water.
In addition, the matrix components of the reaction system can also interact with the sweetener compounds, which can directly or indirectly affect the flavor expression of the sweetener.In addition, as the amount of substrate retention changes, the substrate composition of the wine becomes more complex, including polysaccharides, polyphenols, and proteins, changing the aroma characteristics of the wine [22].In this study, direct dilution followed by injection was proposed, and 1.0 mL of CRW blank matrix was added to the standard working solution to prepare a standard series of solutions.Then, the solution was removed and injected into the instrument for detection.Multiple reaction monitoring (MRM)-Information-dependent acquisition (IDA)-Enhanced product ion (EPI) composite mode acquisition of QTRAP mass spectrometry was applied to obtain MRM data as well as high-sensitivity secondary fragmentation full-spectrum data simultaneously to establish a standard library of nine CRW sweeteners, which was automatically compared with the library of standards to achieve accurate qualitative and MRM data peak area (external standard method) quantitative.Compared with the MRM mode, the EPI spectra were collected by ion trap with enhanced secondary fragmentation subion scanning, which improved the sensitivity, and were full-scan mass spectra with richer confirmatory information, which can effectively assist the qualitative confirmation of samples at trace level or detection limit concentration and make up for the qualitative difficulties of the traditional quadrupole MRM mode for low concentration samples.

Main materials and reagents
Reagents: Experimental water: ultrapure water, prepared by a Milli-Q pure water system.
The Chinese rice wine samples were all commercially available products.

Mass spectrometry conditions
Electrospray ion source; ion source temperature 550°C; electrospray voltage − 4500 V; scan mode: MRM-IDA-EPI; gas curtain gas pressure: 30 psi; nebulizer pressure 55 psi; auxiliary heater pressure: 55.0 psi; collision gas: Medium; polyphenol mass spectrometry conditions are shown in Table 2.When determining the sample, if the retention time of the sample chromatographic peak is consistent with that of the standard, and after deducting the background, the relative abundance of each qualitative ion in the sample spectrogram is compared with the spectrogram of the standard solution obtained under the same conditions with the same concentration to judge the existence of the corresponding analyte in the sample.

Preparation of standard stock solution
Sodium cyclamate, acesulfame, sodium saccharin, sucralose, allyl sweet, neotame, aspartame, stevia extract, and Edelman sweet standard were accurately weighed in a 10 mL volumetric flask, dissolved to scale with ultrapure water to obtain 500 mg/L standard stock solutions, and stored at 4℃.

Optimization of the mobile phase
In this experiment, Kinetex F5 100 A (100 mm×2.1 mm×2.6 μm) was used as the column for the separation and collection of sweeteners into samples.The mobile phase systems of 2 mmol/L, 5 mmol/L, and 10 mmol/L ammonium acetate aqueous solution-methanol were compared, and it was found that the response values of sweetener, acesulfame, and sodium saccharin increased with the increase of ammonium acetate concentration, the response values of allyl sweet remained unchanged, and the response values of neotame, aspartame, stevia extract, and advantame decreased, while the response values of sucralose in the mobile phase systems of 2 mmol/L, 10 mmol/L The mobile phase system of aqueous ammonium acetate solution-methanol showed weak response and bifurcated peak shape, while the peak shape was sharp and better in the mobile phase of aqueous ammonium acetate-methanol at 5 mmol/L.Taking into consideration the mobile phase system of 5 mmol/L ammonium acetate aqueous solutionmethanol was selected, and the MRM chromatograms of the nine sweeteners are shown in Fig. 1 (500 µg/L).

Selection of standard working solutions for substrates
Chinese rice wine [23] is rich in amino acids, vitamins, oligosaccharides, minerals, and other nutrients, so the selection of structural analogs for a blank Chinese rice wine matrix can solve the matrix effect, which can make pretreatment easier and faster and save resources such as consumables.The signal suppression/enhancement (SSE) of the nine sweeteners in Chinese rice wine was investigated by the ratio of the slope of the curve between the ultra-pure water formulation and the 1.0 mL Chinese rice wine matrix formulation.The substrate effect is considered to be insignificant.By comparison, it was found that the substrate aspartame, stevia extract, and advantame standard stock solution in a 10.0 mL volumetric flask, and fix the volume with ultrapure water to the scale to obtain a concentration of 10 mg/L standard intermediate working solution.Pipette 1.0 mL of standard intermediate working solution in a 10.0 mL volumetric flask, and fix the volume to the scale with ultrapure water to obtain the concentration of 1 mg/L standard intermediate working solution.. A 1 mg/L standard intermediate working solution was obtained.Then, 0.10 mL, 0.25 mL, and 0.50 mL of the standard intermediate working solution were pipetted accurately, and 0.10 mL of the standard intermediate working solution was pipetted accurately.Then, 0.10 mL, 0.25 mL and 0.50 mL of standard intermediate working solution were accurately pipetted into a 10.0 mL volumetric flask.In a 10.0 mL volumetric flask, 1.0 mL of each Chinese rice wine blank substrate was added, and the volume was fixed to scale with ultrapure water to obtain standard working solution concentrations of 10, 25, 50, 100, 250, and 500 µg/L.

Sample processing
The sample was accurately weighed to 1.0 g of Chinese rice wine in a 10.0 mL volumetric flask, fixed to the scale with ultrapure water, and fed through a 0.22 μm filter membrane.

Statistical analysis
The mean values and standard deviations of responses obtained by replicate analyses were calculated using the trial version of GraphPad Prism 9 (GraphPad Software Inc., 2236 Avenida De La Playa La Jolla, CA, USA).Other calculations were performed using Microsoft Excel 2019 (Microsoft Corp., Albuquerque, NM, USA).aspartame, stevia extract, and advantame, indicating that substrate effects were not significant [24].The MRM chromatograms of the same concentration of solvent-labeled and matrix-labeled sweeteners are shown in Fig. 2 (100 µg/L).
The sugar content varies greatly among different Chinese rice wines, and according to the sugar content [25], effect was significantly reduced for sweetener, acesulfame, and sodium saccharin, with SSE values of 37% for sweetener, 8.5% for acesulfame, and 57% for sodium saccharin, and the substrate effect was enhanced for sucralose and allyl sweet, with SSE values of 165% for sucralose and 195% for allyl sweet, and SSE values of 80-120% for neotame, not obvious and negligible, therefore, the blank Chinese rice wine matrix was easily obtained and could effectively solve the matrix effects brought about during the detection of Chinese rice wine sweeteners [26].

Establishment of secondary mass spectra of sweeteners
In this experiment, the QTRAP mass spectrometry system with MRM-IDA-EPI scanning mode was used to solve the problem of false positives caused by the complex matrix of Chinese rice wine, and the secondary mass spectra of Chinese rice wine sweeteners were established by the matrix preparation standard solution.the EPI mode, i.e., enhanced subion scanning mode, can obtain higher sensitivity and they can be classified as dry Chinese rice wine (≤ 15.0 g/L), semidry Chinese rice wine (15.1 ~ 40.0 g/L), semisweet Chinese rice wine (40.1 ~ 100.0 g/L), and sweet Chinese rice wine (> 100.0 g/L).In this experiment, one each of the four types of Chinese rice wine was selected for total sugar testing, and their total sugar contents were 2.3, 28.2, 54.0, and 150.0 g/L, which met the total sugar requirements of their four types of Chinese rice wines.The matrix effects of the four types of Chinese rice wines were studied and found to be equivalent for different types of Chinese rice wines.MRM chromatograms of the four types of Chinese rice wines See Fig. 3 (250 µg/L), and by comparing their matrix effects SSE, the SSE values of their nine sweeteners were all in the range of 80-120%, indicating that the matrix effects of different types of Chinese rice wine were Through automatic comparison with the spectrum library of the standard, accurate qualitative and MRM data peak area (external standard method) quantitative results were achieved.By comparing the secondary mass spectrometry of sweeteners with the characteristic spectrum library, trace sweeteners can be effectively detected, and false positives can be avoided.secondary fragmentation spectra of the full mass range with different energy fragmentation [27].The software helps in more accurate screening and characterization by automatic comparison with the spectral library of the matrix standard working solution.The secondary mass spectra of the nine sweeteners are shown in Fig. 4. The standard spectrum library of 9 kinds of rice wine sweeteners was established.

Recovery and precision of nine sweeteners in chinese rice wine
Most sweeteners in Chinese rice wine are nonexistent, and blank substrates are easily available, so the spiking test chose commercially available semidry Chinese rice wine as the sample substrate, and three gradient concentrations of low, medium, and high were selected for the spiking test in the range of the standard curve, and the recoveries [29] and precision were calculated [30].The recoveries and precision are shown in Table 4.The results show that the method has good recoveries and precision.

Qualitative and quantitative results
The method was used to determine the sweeteners in 30 samples of commercially available Chinese rice wine.The results showed that two of the Chinese rice wines tested had sweetener detection, one had sucralose detection and one had neotame detection.According to the software results, the retention time and secondary fragment ion ratio of the samples met the requirements, and the purity score of the measured secondary spectra matched with the database was over 90, reflecting the EPI advantage of QTRAP mass spectrometry [31], which can still obtain highly sensitive secondary fragment spectra for low concentration compounds in complex Chinese rice wine matrices, helping to better discriminate false positives and false negatives and ensure accurate qualitative results.Quantification with MRM data showed that the measured Chinese rice wine was detected at 0.424 mg/kg and 0.984 mg/kg for sweetener, 0.184 mg/kg for sucralose, and 0.326 mg/kg for neotame.

Linearity range, method detection limit, and limit of quantification
The method limits of detection and limits of quantification for the nine sweeteners were calculated by fitting the standard curves with the signal-to-noise ratios S/N ≥ 3 and S/N ≥ 10.As shown in Table 3, the correlation coefficients were all greater than 0.997, indicating that the mass of each control correlated well with the peak areas within this linear range [28].The results showed that the method was accurate

Fig. 4
Fig. 4 Secondary mass spectra of nine sweeteners

Table 1
Gradient elution conditions of sweeteners