Rapid determination of 48 stimulant residues in milk and dairy products by high-performance liquid chromatography coupled with quadrupole/Orbitrap high-resolution mass spectrometry

A fast and easy method was proposed for the determination and quantification of 48 stimulants in milk and dairy products using high-performance liquid chromatography coupled with quadrupole/electrostatic field Orbitrap high-resolution mass spectrometry (Q Exactive). The samples were extracted with acetonitrile having 0.1% formic acid, and then, the extracting solution was purified using a Captive EMR-LIPID solid-phase extraction column. The main force between the target compound and the adsorbent was size exclusion. Unbranched long aliphatic chains on the lipids entered the sorbent, while bulky analytes did not, which could effectively remove the phosphorus esters in dairy products. Finally, we determined the instrument conditions. The samples were separated on a C18 column and eluted with acetonitrile and 0.05% formic acid aqueous solution as the mobile phases. Determination was then performed by mass spectrometry in electrospray ion source mode and monitored in parallel by reaction monitoring full scan/data-dependent secondary scan (Full MS/DD-MS2) mode. Under optimal conditions, the limit of quantification for the 48 stimulants was 0.5–100 µg L−1 in the milk and dairy products samples. The recoveries were 60.6–118.9% for milk, 63.2–123.8% for milk powder, and 61.8–119.6% for yogurt, with a determined coefficient of variation of less than 15%. The limit of detection values was 0.1–5.0 µg kg−1, and the limit of quantification values for the analytes was 0.2–10.0 µg kg−1. Finally, this method was used to screen 48 stimulants in 50 milk and dairy product samples.


Introduction
Milk and dairy products are some of the main sources of nutritional meal protein for athletes. However, due to the unreasonable use of stimulants, food-derived stimulant residues have been found in milk and dairy products, which are banned drugs stipulated by international sports organizations.
Stimulants mainly consist of β-agonists, β-blockers, protein assimilating agents, glucocorticoids, steroids, and diuretics (WADA2005; WADA2016). In livestock breeding, the misuse and abuse of stimulant drugs have been reported, such as protein assimilating agents and hormones. As a result, stimulant drug residues may be present in milk and dairy products, and the possibility of illegally added stimulant drugs to milk and dairy products cannot be ruled out. On the eve of the 2008 Beijing Olympic Games, Ouyang Kunpeng, the most likely candidate to win a medal in the Chinese men's swimming team, was banned for life when his urine tested positive after he ate a kebab containing clenbuterol with a friend when he went home on vacation. Among all foodborne stimulants, clenbuterol is the most difficult to prevent. In actual testing, the proportion and content of clenbuterol in meat food on the market is much higher than the safety standards for athlete meat food, while the positive proportions of pork, beef, mutton, and viscera in meat food are higher than in other meat. To uphold the competition regulations and prevent health complications from the consumption of contaminated milk and dairy products (Zhao et al. 2018), a method to rapidly detect stimulant drug residues in milk and dairy products is urgently needed. To meet the relevant requirements of doping detection in various competitions, the analysis must be fast. However, most existing doping detection methods are limited by the types of stimulants they detect, having low detection efficiencies and long analysis times. In this study, liquid chromatography-high-resolution mass spectrometry detection was used to rapidly screen and quantitatively analyze 48 stimulants in 6 categories. This detection technology greatly improved detection efficiency.
The quick, easy, cheap, effective, rugged, safe (QuECh-ERS) method and solid-phase extraction have typically been used for sample pretreatment (Meng 2021). For the determination of 46 stimulant residues in pork, beef, mutton, eggs, and milk, Zhang et al. used a mixed cation exchange solid-phase extraction column for purification using the QuEChERS method. Various β-stimulant veterinary drug residues were detected, and a solid-phase extraction column was sufficient for purification. However, purification steps such as activation, sample loading, rinsing, and elution, prolonged the analysis time. In another study, five compounds in milk, including ciprofloxacin, were determined using on-line solid-phase extraction (Wilde et al. 2018). The method afforded rapid, online sample clean up, rapid analysis, and minimal solvent use, but it required a dedicated Turbo Flow instrument to connect to a liquid pump. Therefore, this method was not universal. A Captive EMR-LIPID pass-through solid-phase extraction column was used in this experiment. Compared to other solid-phase extraction columns, the purification time was fast and interferences, such as protein, fat, and phospholipids in the milk and dairy products, were effectively removed.
At present, only a few studies have reported on the simultaneous determination of limited stimulants in food, and the detection methods for stimulants include gas chromatography (GC) (Wu 2021;Lenka et al. 2021), gas chromatography-mass spectrometry/mass spectrometry (GC-MS/MS) Brailsford et al. 2021;Ahmadkhaniha et al. 2010;Zeng et al. 2010;Dahmani et al. 2018;Azzouz et al. 2011), liquid chromatography (LC) (An 2008), and liquid chromatographymass spectrometry/mass spectrometry (LC-MS/MS) (Wilde et al. 2018;Gilles et al. 2021;Wang et al. 2020;Dorota et al. 2018;Ina et al. 2021;Giovanni et al. 2021;López-García et al. 2018;Guo et al. 2022), time-of-flight mass spectrometry (Q TOF) (Li et al. 2017;Galano et al. 2012;Wei et al. 2021;Liu et al. 2021;Panagiotis et al. 2021;You et al. 2021) and electrostatic field Orbitrap mass spectrometry (Nest et al. 2021;An et al. 2021;Meng et al. 2021;Xu et al. 2021;Kateřina et al. 2021;Yan et al. 2021). According to previous studies, LC-MS has been widely used for the detection of stimulants . As indicated in the literature, although the LC-MS/MS method can be used to analyze stimulants in food, it has only been validated for a limited number of compounds. High-resolution mass spectrometry has the advantages of obtaining first-order accurate mass numbers, isotope ratio information, more complete compound fragmentation and fragment information, and high scanning speed, which can greatly improve the accuracy of qualitative screening. It can be widely used in screening unknown substances in samples and in high-throughput multi-component detection in combination with the database spectrum library retrieval function, but the LC-MS/ MS method cannot be used directly for large-scale screening purposes. In addition, Zhang Haichao et al. reported on the simultaneous determination of 46 foodborne stimulants in foods by ultra-high-performance liquid chromatography-tandem mass spectrometry. However, triple quadrupole tandem mass spectrometry was shown to be limited by matrix interferences for more complex samples. Therefore, the number of target compounds that can be detected simultaneously is limited and has a number of disadvantages, such as false positives. High-resolution mass spectrometry is better suited for complex matrices, as it is more accurate, qualitative, and does not rely on reference material. Thus, accurate screening can be conducted without standards, making it ideal for high-throughput screening and the quantitative determination of multiple stimulants.
This study used Q Exactive electrostatic field Orbitrap mass spectrometry as a rapid and accurate qualitative and quantitative method for detecting veterinary stimulants in milk and dairy products. A total of 48 stimulants, including 16 beta-agonists, 13 anabolic agents, 9 glucocorticoids, and 10 diuretics, were used for rapid qualitative detection and quantitative confirmation.

Reagents and chemicals
Forty-eight stimulant standards (purity ≥ 95%) were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany) and BePure (Beijing, China), and their molecular formulas and Chemical Abstracts Service (CAS) numbers are summarized in Table 1

Samples
The milk consisted of 250 mL of whole milk and skimmed milk packed in a jug. The milk powder consisted of 10,000 g of whole milk powder packed in a bag. The skimmed milk powder was also packed in a bag, and had a weight of 300 g. The infant formula milk consisted of powder stage I (0-6 months) and stage III (12-36 months), and the yogurt consisted of stirred yogurt packed in a cup, with a weight of 180 g. The coagulated yogurt was also packed in a cup and had a weight of 140 g. All samples were purchased from local supermarkets, and the milk powders were stored at room temperature. The milk and yogurt were stored at 0-4 °C. The yogurt was weighed using the wet weight method.

Preparation of the calibration curves
An appropriate amount of each stimulant standard was accurately weighed, and a standard stock solution with a concentration of 1000 µg mL −1 in methanol was prepared, which was stored at − 18 °C and kept in the dark and then, saved for 6 months. The stock solution was diluted with methanol to a concentration of 1 µg mL −1 . An isotopic internal standard stock solution with a concentration of 100 µg mL −1 in methanol was prepared and diluted with methanol to a concentration of 1 µg mL −1 . The intermediate solution was frozen at − 18 °C in the dark for 3 months. The standards at gradient levels were prepared for the calibration curves with progressive dilution of the stock solution of the mixed standard, while the internal standards remained at a fixed concentration (e.g., 10 ng mL −1 ).

Sample extraction and purification
Each sample (2 g) was weighed using a 50 mL polypropylene centrifuge tube, and we then added 20 µL of 1.0 µg mL −1 mixed isotopic internal standard intermediate solution (adrenaline-D 6 , 100 µg mL −1 ). The solution was vortexed to mix evenly. Then, we added 10.0 mL of 1% formic acid in acetonitrile solution to the solution, and the mixture was shaken for 5 min, followed by centrifugation at 4,500 r/min for 5 min (4 °C). Five milliliters of supernatant were transferred to a Captiva EMR-LIPID filtration cartridge (5190-1004), and the filtrate was collected by gravity. After loading, the pressure was appropriately increased to ensure that the flow rate was about 1 drop/s, and the speed was not excessively fast. Then, 2 mL of acetonitrile was used to wash the cartridge, and the unbound and washing fractions were combined. The eluate was dried under a nitrogen stream at 40 °C until nearly dry. Then, the extracts were diluted up to a total volume of 1.00 mL of 0.05% formic acid in water/ acetonitrile (1:1, v/v) for ultrasonic redissolution (20 min, 35 kHz, 40 °C). Finally, the solution was filtered (0.22 μm nylon filter) and transferred to a sample bottle for Q Exactive analysis. The sample was analyzed using primary quantification and secondary mass spectrometry in the Full MS/ DD-MS 2 scanning mode, and no target compounds were found in the tested sample.
The QE MS parameters were as follows: Full MS/ DD-MS 2 , where the quality analyzer consisted of a Q Exactive Orbitrap with a heated electrospray ion source (HESI-II), the capillary temperature was 320 °C, the ion transfer tube temperature was 300 °C, and the spray voltage was 3.5 kV (ESI +) and 2.5 kV (ESI −). The flow rate of the sheath gas (nitrogen) was 4.58 L/min, and the flow rate of the auxiliary gas (nitrogen) was 5.08 L/min. Target SIM-MS 2 mode of the positive and negative simultaneous scans was adopted. The resolution of the full scan was 17,500, and the scanning range was m/z 150-500. Table 1 shows the accurate mass number of the extracted compounds. The full scanning resolution of the primary mass spectra was r = 70,000. The C-trap maximum trap capacity (AGC target) was 5 × 10 5 , and the maximum injection time of the C-trap was 100 ms. The data depended on the secondary ion full scan (Full MS/DD-MS 2 ), with a resolution of 17,500. The C-trap maximum capacity (AGC target) was 5 × 10 4 , where the maximum injection time of the C-trap was 50 ms. The dynamic exclusion time was 5 s.

Optimization of the mass spectrometry conditions
This study adopted two scanning modes for optimization: the first was parallel response monitoring (PRM), and the second was the full MS data-dependent MS 2 (Full MS/ DD-MS 2 ) mode for the full scan data of the high-resolution mass spectra. The experimental findings showed that PRM mode required no more than eight target compounds in the same time period; otherwise, the scanning points were not sufficient. The chemical properties of the target compounds were similar, with the same properties as their internal standard compounds. As a result, their retention times were similar, the use of PRM mode would lead to insufficient scanning points for some compounds, and thus, the scanning mode of Full MS/DD-MS 2 was adopted in this study. The Q Exactive Orbitrap/MS was operated in Full MS/DD-MS 2 scanning mode. Figures. 1, 2, 3 and  4 show the ion chromatograms of the standard solutions of each target compound.
The full-scanning of mass spectra was carried out in simultaneous positive and negative ion mode. Most of the target compounds of the stimulants were [M + H] + or [M + H] − molecular ion peaks. However, the [M + H] + peak of spironolactone was not as high as its [M-C 2 H 3 OS] + response value after removing an acetylthiol group, which was the same as that of the canisterone parent ion. The [M-COOH] − peak of cortisone and prednisolone was higher than the [M + H] + peak. However, in high-resolution mass spectrometry, the responses of the two ion peaks were similar, and the [M + H] + peak was more stable, which was possibly due to a high energy source of ESI (±), and the ion peak that formed by the combination of ion fragments and COOH − was more stable, while QE consisted of full mass scanning with low energy and the H + peak was more stable.

Resolution selection
The biggest advantage of high-resolution mass spectrometry was its high resolution, which allowed for quantification of the primary mass spectra and a reduction in sample matrix interference. In general, different resolutions indicated different anti-interference abilities of the target compounds. First, 2.0 µg/kg atenolol was added to the blank milk matrix, and then, the anti-interference abilities of the target compounds with resolutions of 70,000, 17,500, and 35,000 were investigated. Compared to the extracted ion chromatograms, the interference of target compounds could be effectively removed under the condition of a resolution of 70,000, and the chromatograms of all target compounds were clear and complete. Therefore, a full mass resolution of 70,000 was selected for analysis.

Quality accuracy
The first-order mass number extracted by high-resolution mass spectrometry was accurate to three decimal places, and the accurate mass numbers of first-order mass spectrometry were qualitative, so it was necessary to select an appropriate resolution to improve the accuracy of qualitative and quantitative analysis. The experiment showed that when the resolution reached 70,000, the target compound and interfering substance were completely separated from the baseline, which could effectively remove matrix interference. We thus realized the accurate qualitative and quantitative analysis of the target compounds. As shown in Table 1, the mass accuracy of the target compound was high and could meet the qualitative requirements.

Optimization of the secondary mass spectrometry conditions and establishment of a spectrum library
The data-dependent secondary mass spectrometry scanning (Full MS/DD-MS 2 ) dynamic exclusion mode was adopted. When the parent ion in the list appeared and the strength reached the set threshold, the secondary data were automatically collected. Dynamic exclusion ensured that low content of common components obtained the Full MS/ DD-MS 2 fragment spectrum. We could optimize the collision energy of the parent ion. In the mass spectrum, the abscissa was the mass-nucleus ratio and the ordinate was the ion strength, which was generally expressed by relative abundance, where the strongest peak (the highest abundance of the child ion) was 100%. Then, the fragment-rich secondary spectra were obtained for qualitative confirmation of the target compounds.

Selection of chromatographic column
The target compounds were all weakly polar compounds. According to the principle of similar phase solubility, the C 18 column was suitable for separating non-polar or weakly polar compounds, along with a Waters BEH C 18 column (100 mm × 2.1 mm, 1.8 μm), Waters HSS T3 column (100 mm × 2.1 mm, 1.8 μm), and a CAPCELL PAK C 18 (2.0 mm × 150 mm, 5 μm) were used for investigation in the experiment. The results showed that these four C 18 columns from different manufacturers provided good separation of the compounds except isomers. The resolutions of the two isomers of prednisolone and cortisone, canrenone, and spironolactone on the Waters BEH C 18 column were 0.8 and 0.9, respectively, the resolutions on T3 column were 0.7 and 0.8, respectively, and the resolutions on the CAPCELL PAK C 18 column were 1.2 and 1.3, respectively. According to the resolution calculation formula, the calculation formula of the resolution (R) is as follows: where the greater the R, the better the separation of the two adjacent components. Therefore, the CAPCELL PAK C 18 (2.0 mm, 150 mm, 5 μm) chromatographic column was used for further detection.

Selection of LC conditions
A variety of stimulants, including β-agonists, protein assimilating agents, glucocorticoids, and diuretics, were isolated. There were significant differences among the categories, and they contained three pairs of isomers: canrenone and spironolactone, dexamethasone and betamethasone, and prednisolone and cortisone. According to the principle of similar phase dissolution, to obtain better separation efficiency and a sharp peak shape, 0.05% formic acid in water, 0.1% formic acid in water, and 2 mmol/L of R = 2 t R 2 − t R 1 W 1 + W 2 , ammonium acetate were compared. The addition of formic acid improved the ionization rate of the target compounds. When the mobile phase was 0.1% formic acid in water, glucocorticoids, such as prednisone, were ionized by complexing −COOH groups. When the concentration of formic acid in water was reduced to 0.05%, glucocorticoids were ionized into a stable −H peak. Additionally, the peak shape improved, and the tailing phenomenon of adrenaline, ractopamine, and the other compounds with short retention times was reduced. The addition of ammonium acetate had no obvious effect on the improvement of peak shape.
To simplify the method and stabilize the peaks, 0.05% formic acid in water was selected as the mobile phase, and acetonitrile and methanol were compared as the organic mobile phase. When methanol was used, the target compounds in the matrix standard overlapped, and the peaks of adrenaline and the other compounds exhibited tailing. By contrast, when acetonitrile was used, tailing was reduced. Therefore, acetonitrile and 0.05% formic acid in water were used as the mobile phases for gradient elution.

Selection of extraction solvent
The stimulants selected in this study covered an extremely wide polarity range. For example, the beta-agonists were weakly basic polar compounds, while the anabolic hormones had good solubility in weakly or moderately polar solvents, and the glucocorticoids were highly hydrophobic. Most diuretics contain both acidic and alkaline groups with very different properties. Therefore, the selection of an appropriate extraction solvent was very important. Methanol, methanol containing 0.1% formic acid, acetonitrile, and acetonitrile containing 0.1% formic acid were selected to investigate the extraction efficiency by standard addition recovery. When the 48 stimulants were extracted with methanol and methanol containing 1% formic acid, the extract was turbid when it passed through the purification column, and the recovery rates of zelenol, beclomethasone, acetazolamide, and the other compounds were as high as 200%. The purification effect was poor, and the matrix interference of the target compound was significant. When acetonitrile was used as the extraction solvent, the recovery rate was 70%-120%. Acetonitrile could precipitate protein and fat in the sample matrix, but the recovery of adrenaline was as low as 20%, and that of hydrochlorothiazide was as high as 300%. When the extract was 0.1% acetonitrile formate, the recovery was 60-120%. Thus, 0.1% acetonitrile formate was selected as the extraction solvent in this experiment.

Selection of a purification column
The purification efficiencies of the three different purification methods were investigated. The first method involved omitting the purification step and directly loading the sample after sample extraction. The recovery of all stimulants was low (less than 60%), and the recovery of protein assimilating agents, such as norone propionate, was less than 20%. Although acetonitrile, as the extraction solvent, could precipitate protein, the phospholipids in milk interfered with analyte recovery. When a prime HLB column was employed (PRiME HLB, a new type of reversed-phase solid phase extraction (SPE) adsorbent with a fast-passing solid phase extraction column that omits the conventional activation, leaching, and elution steps), the recoveries of fenoterol, simatro, noraconitine, and zipatero were less than 20%. When the Captiva EMR-LIPID purification column was used, because the main force between the target compound and the adsorbent was size exclusion, unbranched long aliphatic chains on the lipids entered the sorbent while bulky analytes did not, which could effectively remove the phosphorus esters in dairy products. Moreover, good recoveries of each target compound were obtained, and the matrix inhibitory effects of salbutamol, terbuterol, testosterone propionate, and chlorothiazone were significantly reduced. In addition, the interference peak near the atenolol peak was eliminated. Thus, the Capture EMR-LIPID solid-phase extraction column was selected for purification.

Selection of the reconcentrated solution
The effluent was collected, blow-dried under nitrogen, and a solvent was chosen to dissolve the reconcentrated solution.
The experiment compared the reconstitution of acetonitrile with two reconstitution solutions, namely, 0.05% formic acid in water/acetonitrile (9:1, v/v) and 0.05% formic acid in water/acetonitrile (1:1, v/v). The results showed that when using 0.05% formic acid in water/acetonitrile (9:1, v/v), the recovery of norone propionate and norone phenylpropionate was as low as 30%, possibly because the two compounds were almost insoluble in water. Using 0.05% formic acid in water/acetonitrile (1:1, v/v), the recoveries of norone propionate and norone benzoate significantly improved and met the 20 µg kg −1 quantitative requirements. Therefore, 0.05% formic acid in water/acetonitrile (1:1, v/v) was selected as the reconcentrated solution.

Effect of nitrogen drying
Target compounds, such as adrenaline, were greatly affected by the nitrogen drying temperature and duration. When the extract was directly loaded after purification, the recovery rate of adrenaline was about 100%, but the recovery rates of salbutamol and testosterone propionate were less than 60%. This was possibly because the extract was not concentrated, resulting in dilution of the target compounds and a reduction in sensitivity. When the nitrogen stream temperature was lower than 40 °C and nitrogen was blown until nearly dry, the extracts were diluted up to 1.0 mL with 0.05% formic acid in water/acetonitrile (1:1, v/v). The recovery rate of adrenaline was approximately 100%. However, when the nitrogen stream temperature was too high, the recovery rate was less than 20%. Therefore, adrenaline was greatly affected by temperature. When using the CAPTIVA degreasing column for purification and filtration, the nitrogen stream temperature and duration needed to be considered.

Evaluation of the matrix interference
Matrix effects are a common problem in mass spectrometry. Matrix effects can enhance or weaken the response value of the target, thus, affecting the accuracy and selectivity of the detection results: where A is the response value of target compound in a pure solvent, and B is the response value of the target compound with the same content added in the sample. Taking infant formula milk powder as an example, the matrix effect of noraconitine was 75%, that of terbutaline was 80%, that of fenoterol was 85%, that of atenolol was 81%, that of hydrocortisone was 79%, that of methylprednisolone was 78%, and that of beclomethasone was 75%. The matrix effect of such target compounds will reduce the response value of the target compound. To reduce the matrix effect and obtain more accurate quantitative results of hydrochlorothiazide, Matrix effect (%) = B∕A × 100, the target compounds were quantitatively analyzed using blank matrix labeling and an isotopic internal standard.
In the experiment, we found that the recovery value of hydrochlorothiazide-D 2 as the internal standard was always low when detected using UPLC-MS/MS, and the larger the scalar amount, the more significant the impact on the spiking recovery value. When detected using a Q Exactive mass spectrometer, this phenomenon was significantly improved. The reason for this was that the chlorine element (Cl) contained in hydrochlorothiazide had two main isotopes, 35Cl and 37Cl, and their relative atomic masses were 34.96885 and 36.96590, respectively, with an abundance ratio of about 100:32. As a result, there were two isotopic ion peaks of m/z 295.95745 and 297.95398 in hydrochlorothiazide during mass spectrometry detection, while the mass-to-charge ratio of the internal standard of hydrochlorothiazide-D 2 used in the experiment was m/z 297.96948. During detection, the 37Cl isotope peak of hydrochlorothiazide and the internal standard hydrochlorothiazide-D 2 could not be extracted and separated by the mass-to-charge ratio of UPLC-MS/MS, resulting in an abnormal increase in the response value of the internal standard, which affected the quantitative accuracy, where the higher the content, the greater the impact.

Method validation
A blank matrix mixed with standard solutions of mass concentrations of LOQ, 2 LOQ, 5 LOQ were prepared a total of 6 times for each mass concentration, and Table 2 shows the specific standard curve range for determination. The mass concentration (x, µg L −1 ) and the linear regression equations of 48 target compounds were obtained. Fenoterol, demethyl-coclaurine, nandrolone phenylpropionate, and zilpaterol were quantified by the external standard method, and the other target compounds were quantified by the internal standard method. As shown in Table 2, the linear relationship of the 48 stimulant compounds was good, and the correlation coefficient ranged from 0.9912 to 0.9998. The detection limit (LOD) and quantitative limit (LOQ) of the method were defined as the concentrations when the quantitative ion signal-to-noise ratios were 3 and 10, and the values for each target compound are shown in Table 2. Blank milk, yogurt, and milk powder samples were used as the matrices, 48 standard solutions of stimulant compounds were spiked into each matrix, where the spiked levels were (LOQ), 2 LOQ, and 5 LOQ, respectively. Six samples were measured in parallel for each spiked level, and the results are shown in Table 2. The recoveries of 48 stimulant compounds ranged from 60.6 to 123.8%, and the RSD values were 1.3-10.0%, with a determined coefficient of variation of less than 15%, thus meeting the requirements for quantitative determination.

Actual samples analysis
The established method was used to screen 43 batches of milk, yogurt, and milk powder purchased from the supermarket. Among these, 20 batches of infant milk powder, 15 batches of yogurt, and 18 batches of milk powder were not analyzed for 48 stimulant residues. Since significant international attention has been paid to food safety, especially in infant food, this study provided technical support for the rapid screening of stimulants in infant food.

Conclusions
High-performance liquid chromatography coupled with quadrupole/electrostatic field orbital trap high-resolution mass spectrometry was used to establish a rapid qualitative screening and quantitative analysis method for 48 stimulant residues in milk and dairy products. The method showed good performance in terms of recovery, precision, accuracy, MDL, and MQL, proving the effectiveness of the methodology for analysis of these compounds. Compared to traditional methods, this method reduced matrix interference, greatly improved the accuracy of the qualitative and quantitative analyses, and enhanced the sensitivity, resulting in an effective method for detecting stimulants in milk and dairy products.  Intra-day (RSD, n = 3) Intra-day (RSD, n = 6) LOD (µg kg −1 ) LOQ (µg/kg −1 ) Determination coefficient (R 2 ) of Xi'an customs P. R. China (Grant No. K-202102), and General Administration of Customs Science and Technology Program (Grant No. 2021HK209).

Data availability
The data sets supporting the results of this article are included within the article and its additional files.

Conflict of interests
The authors declared that they have no competing interests.