Method for screening illegal additives of weight loss food based on high resolution mass spectrometry

Abstract

The application belongs to the technical field of food safety detection, and specifically discloses a method for screening illegal additives in weight-reducing food based on high-resolution mass spectrometry. The method comprises the following steps: obtaining a sample, sample pretreatment, mixing standard working solution preparation, database screening setting and actual sample screening determination. If the relative deviation of the retention time of the to-be-tested compound in the sample solution and the retention time in the spectrum library and the signal-to-noise ratio of the monitored qualitative ions exceed the threshold value, the relative deviation of the accurate mass number of the parent ion and the theoretical mass number exceeds the threshold value, and the relative deviation of the accurate mass number of a secondary fragment ion exceeds the threshold value, it can be preliminarily judged that the compound is contained in the experiment. After the sample is ultrasonically extracted by 80% methanol water, centrifuged, degreased and purified, and then filtered through a 0.22 mu m filter membrane, the sample is separated by a chromatographic column and then fully scanned by high-resolution mass spectrometry, so that qualitative and quantitative analysis can be performed on the database of 67 kinds of illegal additives, high-sensitivity and high-accuracy screening of multiple categories of illegal additives can be realized, and the method is suitable for weight-reducing food quality supervision.

Description

Technical Field

[0001] This invention belongs to the field of food safety testing technology, specifically relating to a method for screening illegal additives in weight-loss foods based on high-resolution mass spectrometry (HRMS). Background Technology

[0002] In recent years, the demand for health food products has been increasing, especially for weight-loss products. However, to achieve the claimed effects, many unscrupulous merchants are illegally adding chemical drugs to weight-loss products, including appetite suppressants (sibutramine), laxatives (phenolphthalein, bisacodyl), hypoglycemic drugs (metformin), and diuretics (furosemide). These substances may pose serious health risks such as cardiovascular disease and liver and kidney damage. Traditional detection methods, such as high-performance liquid chromatography (HPLC) or gas chromatography-mass spectrometry (GC-MS), have limitations such as low throughput, high false positive rate, and poor matrix suitability. In recent years, screening technologies based on high-resolution mass spectrometry (such as UHPLC-Orbitrap HRMS) have been gradually applied. For example, the method reported by Xu Hongbin et al. achieved the screening of 32 illegal additives within 17 minutes using Orbitrap high-resolution mass spectrometry. However, this method still has the following significant drawbacks: The scope of target substances is insufficient: it does not cover novel derivatives (such as bisacodyl and norchlorocalceline) and multiple categories of drugs (such as glucocorticoids and thyroid hormones), which cannot meet the regulatory needs of the current diversification and complexity of illegal additives in weight loss foods; The ability to identify isomers is lacking: relying on a single precise mass number matching, without establishing a characteristic fragment ion library and retention time locking (RTL) technology, it cannot effectively distinguish isomers such as phenolphthalein and bisacodyl (both m / z 318.1312), and there is a risk of misjudgment; Limitations in detection efficiency and sensitivity: The single analysis time is as long as 25 minutes, and the ability to detect trace additives (such as sibutramine metabolites <10 ng / mL) is insufficient (LOD is 0.5 μg / mL), making it difficult to meet the screening requirements for trace illegal additives in national standards.

[0003] Therefore, there is an urgent need to develop an efficient, accurate, and scalable screening technology to solve key problems in existing methods, such as narrow detection range, severe matrix interference, high false positive rate of isomers, and low analysis efficiency. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for screening illegal additives in weight-loss foods based on high-resolution mass spectrometry. By establishing a high-resolution mass spectrometry database containing 67 illegal additives and optimizing the pretreatment process to make it applicable to complex matrices such as tablets, capsules, and powders, and by combining isotope peak matching, retention time locking (RTL), and fragment ion library retrieval, efficient and accurate screening of various illegal additives in weight-loss foods can be achieved.

[0005] To address the above technical problems, the present invention provides a method for screening illegal additives in weight-loss foods based on high-resolution mass spectrometry, comprising the following steps: The steps include the following: (1) Sample pretreatment: Sample extraction and purification: The extraction solvent was added to the homogenized sample, and then the sample was mixed, extracted by ultrasound and centrifuged, and then filtered through a 0.22 μm organic filter membrane for instrumental analysis. (2) Preparation of mixed standard working solution: Transfer the corresponding volume of the stock solution of 67 illegally added standards (100 μg / mL) into a 10 mL volumetric flask, dilute to volume with methanol, and prepare a mixed standard solution for sample detection and filter membrane instrument determination; (3) Construct a database screening model: In full MS-ddMS2 Scan mode, high performance liquid chromatography-electrostatic field orbital hydrazine high resolution mass spectrometry was used to perform a first-level mass spectrometry full scan on the target compounds of 10 μg / L standard solutions of 67 illegally added drugs. The molecular weight, retention time and fragment ion information of the 67 illegally added drugs were obtained by high resolution mass spectrometry, thereby establishing a chromatographic library and a first-level standard spectral library of mass spectra of the 67 illegally added drugs. (4) Compound screening conditions for actual samples: Referring to the high-resolution mass spectrometry confirmatory criteria for chemical residues proposed by the US FDA Food and Veterinary Drugs Steering Committee (OFVM), the following conditions must be met in full scan and secondary scan modes to be considered a suspected positive result: extraction must be performed within a 5 ppm mass window: ① Target analyte signal response S / N > 3. If S / N is not present, at least 5 consecutive scan points are required to confirm a single signal; ② The retention time of the target analyte deviates from the retention time parameter in the mass spectrometry library by ≤ 0.2 min or ±2.5% (not exceeding 0.5 min); ③ Compared with the mass spectrometry library, the target analyte can match a primary precursor ion and a secondary fragment ion, with a primary precursor ion mass accuracy deviation < 5 ppm and a secondary fragment ion mass accuracy deviation < 10 ppm. If the substance detected in the sample meets all three conditions after comparison with the mass spectrometry library, it can be considered a positive sample.

[0006] In the optimized scheme, the extraction solvent is 80% methanol-water (containing 0.1% formic acid).

[0007] In the further optimized scheme, the power of the initial ultrasonic extraction was 200-300 W, the frequency was 30-50 kHz, and the extraction time was 10-20 min; the centrifugation speed was 5000-8000 r / min, and the centrifugation time was 3-10 min.

[0008] The final extraction conditions were: extraction power of 300 W, frequency of 400 kHz, extraction time of 15 min; centrifugation speed of 8000 r / min, centrifugation time of 5 min.

[0009] The chromatographic conditions in step (3) are as follows: Waters ACQUITY UPLC HSS T3 column (50 mm × 2.1 mm, 1.8 μm); mobile phase A is 0.1% formic acid aqueous solution, and B is methanol; flow rate is 0.3 mL / min; injection volume is 2.0 μL; column temperature is 40 ℃; gradient elution program: 0 ~ 1.0 min, 95% A; 1.0 ~ 2.5 min, 95% ~ 5% A; 2.5 ~ 4.0 min, 5% A; 4.0 ~ 4.1 min, 5% ~ 95% A; 4.1 ~ 5.0 min, 95% A.

[0010] In the optimized scheme, the WATERS ACQUITY HSS T3 column was selected, which effectively retains water-soluble and highly polar small molecule compounds, produces good peak shapes, and is beneficial for separating the main interfering components in the matrix, such as sugars and other non-polar substances.

[0011] It was found that most target compounds are more stable under acidic conditions, which is more conducive to ionization. The acid in the mobile phase can provide the H+ required by the ESI source, thereby improving the ionization efficiency, significantly improving the peak shape of the compounds, and increasing the sensitivity. In order to take into account the peak shape and mass spectrometry response of 67 compounds, 0.1% formic acid water methanol was selected as the mobile phase for this experiment.

[0012] The 67 illegally added substances are sibutramine, N,N-bisdemethylsibutramine, homosibutramine, fenfluramine, phenylpropanolamine, amphetamine, benzylsibutramine, cloxibutramine, rimonaban, citalopram, topiramate, sertraline, sibutramine dichloroisoamyl substitute, ephedrine, pseudoephedrine, caffeine, methylephedrine, theophylline, glibenclamide, glibenclamide, gliclazide, glimepiride, glipizide, glimepiride, phenformin, pioglitazone, repaglinide, rosiglitazone, tolbutamide, orlistat, and bumeta. Nitroglycerin, torasemide, eplerenone, amiloride, triamterene, phenolphthalein, bisacodyl, bicyclopropionate, bisacodyl, bisphenol salbutamol, bis(chlorobutyropropionate), 4-chlorobutyropropionate, 4-chlorobisisobutyropropionate, fluoxetine, paroxetine, nefazodone, lovastatin, simvastatin, bezafibrate, fenofibrate, benzocaine, clenbuterol, ractopamine, salbutamol, terbutaline, naltrexone, zonisamide, furosemide, ethacrynic acid (ethacrynic acid), hydrochlorothiazide, chlorthalidone, methaqualone, benzylthiazide, cyclothiazide, probenecid, emodin.

[0013] This invention discloses a method for screening illegal additives in weight-loss foods based on high-resolution mass spectrometry. It utilizes high-performance liquid chromatography-quadrupole / electrostatic field orbital hydrazine high-resolution mass spectrometry, combined with Trace Finder screening software, to simultaneously screen 67 illegal additives in weight-loss foods with a single injection, achieving high-throughput simultaneous screening. Furthermore, the method optimizes the pretreatment conditions to affect the screening compounds, thereby enabling rapid, high-throughput screening and analysis of illegally added drugs in weight-loss foods.

[0014] This invention simplifies the sample pretreatment steps, making the operation simpler and more economical.

[0015] This invention solves the problem of qualitative detection of various illegal additives in foods claiming to have weight-loss effects in the field of food testing. It is simple to operate, time-saving, labor-saving, and has high throughput. It is suitable for testing institutions to conduct daily monitoring of illegal additives in foods claiming to have weight-loss effects. Attached Figure Description

[0016] Figure 1 This is a comparison diagram of the sibutramine positive sample and the standard in this invention; Figures 2-68 The primary extraction ion chromatogram and secondary mass spectrometry of 67 illegally added drugs (100 ng / mL) under full scan plus automatic trigger secondary scan mode. Detailed Implementation

[0017] The present invention will be further described below with reference to specific embodiments: Among them, instruments: Q-Exactive quadrupole-electrostatic track trap high-resolution mass spectrometry system (Thermo Scientific, USA); Vanquish UHPLC ultra-high performance liquid chromatography system (Thermo Scientific, USA); Waters ACQUITY UPLCHSS T3 (50 mm × 2.1 mm, 1.8 μm) liquid chromatography column; VORTEX GENIUS3 vortex mixer (IKA, Germany); Milli-Q ultrapure water system (Millipore, USA); MSE225P-ICE-DU electronic balance (Sartorius, Germany); ultrasonic cleaner (Elma, Germany).

[0018] Standards and reagents: Sixty-seven standards were purchased, including a mixed standard of 24 drugs (BJS201701, TM-81809a, 100 μg / mL), a standard for the determination of 19 diuretics including torasemide in food (BJS 202409, BePure-30688XA, 100 μg / mL), a mixed standard of 17 compounds including bis(acetyl)butanol in food (BJS 202209, BePure-30794CZ, 100 μg / mL) in acetonitrile (containing dimethyl sulfoxide and acetone), and 7 sibutramines (100 μg / mL). Information on the 67 compounds is shown in Table 2. Acetonitrile and methanol (Merck, Germany); formic acid (CNW, Germany) were all chromatographically pure; the experimental water was Milli-Q ultrapure water. Example

[0019] A method for screening illegal additives in weight-loss foods based on high-resolution mass spectrometry was proposed. Sixty-seven representative illegally added drugs were selected. By optimizing pretreatment conditions and combining ultra-high performance liquid chromatography with tandem quadrupole electrostatic field orbital ion trap high-resolution mass spectrometry, high-throughput screening of illegally added drugs in popular weight-loss foods was conducted. The specific steps are as follows: (1) Sample collection: Compressed candy, solid beverage, fruit and vegetable juice beverage, substitute tea, jelly and other samples were collected from various supermarkets and online shopping platforms (Taobao, Douyin, Pinduoduo, Vipshop, Liangjiu, Fengxiangdian, etc.).

[0020] (2) Sample pretreatment: Accurately weigh 1.0 g (accurate to 0.0001 g) of sample powder into a 50 mL stoppered centrifuge tube, add 20 mL of methanol, vortex for 1 min to mix thoroughly, and sonicate for 15 min. Cool to room temperature, centrifuge at 8000 r / min for 5 min, transfer the supernatant to a 50 mL colorimetric tube, and repeat the extraction once more. Combine the two extracts, dilute to the mark with methanol, and mix well. Filter through a 0.22 μm microporous membrane before analysis. Dilute with methanol to a suitable concentration if necessary.

[0021] (3) Solution preparation: Standard stock solutions: Accurately transfer appropriate amounts of each of the 67 weight-loss standard solutions, dissolve them in methanol, and dilute to 10 mL to prepare standard stock solutions with a concentration of 10 μg / mL. Store at -18 ℃ protected from light.

[0022] Mixed standard solution: Accurately pipette appropriate amounts of each of the 81 standard stock solutions and dilute to 10 mL with methanol to prepare a mixed standard solution with a concentration of 1.0 μg / mL. Store at -18 ℃ protected from light.

[0023] Standard working solution: Take an appropriate amount of the mixed standard solution as needed, dilute it with methanol to the required concentration, and use it immediately.

[0024] (4) Liquid chromatography-tandem mass spectrometry conditions: Chromatographic conditions: Waters ACQUITY UPLC HSS T3 column (50 mm × 2.1 mm, 1.8 μm); mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: methanol; flow rate: 0.3 mL / min; injection volume: 5.0 μL; column temperature: 40 ℃; gradient elution program: 0~1.0 min, 95% A; 1.0~2.5 min, 95%~5% A; 2.5~4.0 min, 5% A; 4.0~4.1 min, 5%~95% A; 4.1~5.0 min, 95% A. See Table 1 for details. Table 1. Gradient elution program for mobile phase 0 95 5 1.0 95 5 2.5 5 95 4.0 5 95 4.1 95 5 5.0 95 5 Mass spectrometry conditions: Heated electrospray ionization source (HESI), positive and negative ion switching mode; Detection method: Full scan plus automatic triggering secondary scan (full MS-ddMS) 2 Capillary heating temperature: 320℃; Sheath gas (N2) flow rate: 40 L / min; Auxiliary gas (N2) flow rate: 10 L / min; Purge gas (N2) flow rate: 3 L / min; Spray voltage: 3 kV; Full MS scan resolution (R): 70000; Automatic gain control (AGC target): 3×10⁻⁶6 Maximum dwell time: 100ms; Scan range: m / z 100~1500; Threshold trigger value: 1×10 5 Vertex trigger time: 2~8s; Dynamic exclusion time: 10s; ddMS 2 Scan resolution (R): 17500; Automatic gain control: 1×10⁻⁶ 5 Maximum dwell time: 50ms; TopN: 5; Separation window: m / z 4.0; Collision energy (NCE): 20, 40 and 60 eV respectively; Collision energy step value: 50%.

[0025] (5) Construction of database screening model: Construction of chromatographic and mass spectra libraries of 67 illegally added drugs; In full MS-ddMS 2 In Scan mode, high-performance liquid chromatography-electrostatic field orbital hydrazine high-resolution mass spectrometry (HPLC-ESMMS) was used to perform a first-level full-scan mass spectrometry scan on the standard solutions (10 μg / L) of 67 illegally added drugs. The molecular weight, retention time, and fragment ion information of the 67 illegally added drugs were obtained through high-resolution mass spectrometry, thereby establishing a chromatographic library and a first-level standard library of mass spectra for 68 illegally added drugs. Specifically, a mixed standard solution was prepared and injected according to the established chromatographic and mass spectrometric conditions. The Chinese and English names, CAS numbers, and chemical formulas of 67 illegally added drugs were accurately entered. Trace Finder™ 5.0 (Thermo Fisher Scientific, USA) was used to calculate the theoretical mass number of each compound. Then, data-dependent secondary mass spectrometry (ddMS2) was performed using dynamic exclusion mode. When the response intensity of the precursor ion in the list reached a set threshold, secondary data were automatically acquired. The target analytes were collided at three energies (20 / 40 / 60 eV), and the results were summed to obtain the secondary spectra. The precise molecular weights of the major secondary fragments were calculated using software and correlated with the retention times, precise mass numbers, Chinese and English names, CAS numbers, and molecular formulas of the corresponding compounds. Finally, the spectral library of 67 illegally added drugs was constructed, as shown in Table 2 below. Figures 11-66 As shown.

[0026] Table 2 Information on 67 Types of Illegal Database Additions

[0027] (6) Setting up qualitative methods: According to EU SANTE 11813 / 2017 regulations, qualitative analysis is based on the retention time of a compound, the precise mass number of the precursor ion, and the precise mass number of one fragment ion (or two precise mass numbers of the precursor ions). Under the same conditions, if the relative deviation between the retention time of the analyte in the sample solution and the retention time in the spectral library is ≤±2.5% (and does not exceed 0.5 min) or ≤0.2 min, the signal-to-noise ratio (S / N) of the monitored qualitative ion is ≥3, the relative deviation between the precise mass number of the precursor ion and the theoretical mass number is ≤5 ppm (1×10⁻⁶), and the relative deviation of the precise mass number of one secondary fragment ion is ≤10 ppm, then it can be preliminarily determined that the compound is present in the experiment.

[0028] The target peak area threshold is set to 50,000 to exclude the influence of some impurity peaks and reduce screening time. The signal-to-noise ratio threshold is set to 10, and the exact mass number deviation is set to 5×10⁻⁶. The retention time determination mode is set to confirm, and the retention time window width is 30 s. The fragment ion determination mode is set to confirm, with a minimum matching degree of 1 fragment ion, a fragment ion intensity threshold of 5,000, and a fragment ion mass number deviation of 1×10⁻⁵. Isotope screening can be used as supplementary screening information for the target peak. When enabled, the determination mode is confirmed, the matching degree threshold is 90%, the mass number deviation is 1×10⁻⁵, and the intensity deviation is 20%. Example

[0029] Method verification and validation: 1. Linearity Range and Limit of Detection Validation: Working solutions of mixed reference standards (0.2-400 ng / mL) were tested, and a standard curve was plotted with concentration on the x-axis and peak area ratio on the y-axis. The results showed that the 67 illegally added substances exhibited good linearity within their respective concentration ranges, with correlation coefficients all greater than 0.99. The limits of quantitation (LOQs) were calculated using a signal-to-noise ratio of 10, and the LQs for each compound ranged from 0.01 to 0.40 mg / kg, indicating that this method has high sensitivity.

[0030] 2. Precision Validation: Six parallel experiments were conducted using compressed candy to determine the content of each target compound. The results showed that the relative standard deviation (RSD) of each compound content was between 1.1% and 3.5%, indicating that the method has good precision.

[0031] 3. Accuracy Validation: A spiked recovery experiment was conducted. Mixed control solutions at low, medium, and high levels were added to weight-loss food samples known to be free of the target illegal additive, with each level tested in triplicate. The results showed that the spiked recoveries of each compound ranged from 71.0% to 119.5%, indicating that this method has high accuracy.

[0032] Table 3. Validation results of 67 drugs in compressed candies Example

[0033] Actual sample testing: Fifty batches of commercially available compressed candies (20 herbal, 10 white kidney bean, 10 probiotic, 5 coffee bean, and 5 others) were tested. Illegal additives were detected in 11 batches (6 herbal, 3 white kidney bean, 1 probiotic, and 1 coffee bean), a detection rate of 22%. The others were not detected. The detected components were sibutramine, theophylline, sulfanilamide, hydrochlorothiazide, cyclothiazide, and emodin. The retention time, precise mass-to-charge ratio, and characteristic fragment ions of the detected components were consistent with database information, and the content ranged from 0.142 to 625 mg / kg. The results of typical positive samples are shown in Table 4. The most frequently detected components were sibutramine and sulfanilamide. All of the above components are illegal additives in food and should not be detected in principle, indicating a risk of illegal addition in compressed candies. Relevant departments should strengthen supervision.

[0034] Table 4. Test results of typical positive samples

[0035] The above embodiments / experimental examples are merely illustrative and not intended to limit the implementation methods. Those skilled in the art will recognize that various variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementation methods. However, obvious variations or modifications derived therefrom remain within the scope of this invention.

Claims

1. A method for screening illegal additives in weight-loss foods based on high-resolution mass spectrometry, characterized in that: The steps include the following: (1) Sample pretreatment: Sample extraction and purification: Add extraction solvent to homogenized sample, then mix, extract by ultrasound and centrifuge, filter through membrane and analyze. (2) Preparation of mixed standard working solution: Transfer the corresponding volume of the stock solution of 67 kinds of drug standards into a 10mL volumetric flask, dilute to volume with methanol, prepare a mixed standard solution, and store at low temperature and protected from light; (3) Constructing a database screening model: In full MS-ddMS2 Scan mode, high performance liquid chromatography-electrostatic field orbital hydrazine high resolution mass spectrometry was used to perform a first-level mass spectrometry full scan on the 67 illegally added 10 μg / L standard substance solutions to target compounds. The molecular weight, retention time and fragment ion information of the 67 illegally added substances were obtained by high resolution mass spectrometry, thereby establishing a chromatographic library and a first-level standard spectral library of the 67 illegally added substances and their mass spectra. (4) Mass spectrometry analysis: Accurately measure the intermediate solution of the mixed reference standard, and dilute it stepwise with methanol to prepare a series of working solutions of the mixed reference standard with concentrations ranging from 0.2 to 400 ng / mL; The test solution and a series of mixed reference working solutions were injected separately and detected by high-resolution mass spectrometry. Chromatographic separation was performed using a T3 column with a gradient elution of 0.1% formic acid aqueous solution and methanol as the mobile phase. Mass spectrometry scanning was performed using an electrospray ionization source for simultaneous scanning of positive and negative ions in Full MS / dd-MS² mode. (5) Data processing: A database containing retention times, precise mass-to-charge ratios, and characteristic fragment ions of illegally added substances was established, and the mass spectrometry data of the test samples were qualitatively matched and quantitatively analyzed using software.

2. The method for screening illegal additives in weight-loss foods based on high-resolution mass spectrometry according to claim 1, characterized in that: The extraction solvent is 80% methanol-water (containing 0.1% formic acid).

3. A method for screening illegal additives in weight-loss foods based on high-resolution mass spectrometry according to any one of claims 1-2, characterized in that: The ultrasonic extraction power is 200-300 W, the frequency is 30-50 kHz, and the extraction time is 10-20 min; the centrifugation speed is 5000-8000 r / min, and the centrifugation time is 3-10 min.

4. The method for screening illegal additives in weight-loss foods based on high-resolution mass spectrometry according to claim 3, characterized in that: The ultrasonic extraction was performed at a power of 300 W, a frequency of 400 kHz, and an extraction time of 15 min; the centrifugation speed was 8000 r / min, and the centrifugation time was 5 min.

5. The method for screening illegal additives in weight-loss foods based on high-resolution mass spectrometry according to claim 1, characterized in that: The chromatographic conditions in step (3) are as follows: Waters ACQUITY UPLC HSS T3 column (50 mm × 2.1 mm, 1.8 μm); mobile phase A is 0.1% formic acid aqueous solution, and B is methanol; flow rate is 0.3 mL / min; injection volume is 2.0 μL; column temperature is 40 ℃; gradient elution program: 0 ~ 1.0 min, 95% A; 1.0~2.5 min, 95%~5% A; 2.5~4.0 min, 5% A; 4.0~4.1 min, 5%~95% A; 4.1~5.0 min, 95% A.

6. The method according to claim 7, characterized in that: The mass spectrometry scan has a resolution of 50,000–100,000 FWHM and a mass range of m / z 50–1500. The ion source parameters include a spray voltage of 3.0–4.0 kV, a sheath gas flow rate of 30–50 arb, an auxiliary gas flow rate of 5–15 arb, and a capillary temperature of 300–350 °C.

7. The method according to claim 1, characterized in that: Qualitative matching and quantitative analysis were performed using TraceFinder software.

8. A method for screening illegal additives in weight-loss foods based on high-resolution mass spectrometry, characterized in that: The 67 illegally added substances are sibutramine, N-monodemethylsibutramine, N,N-bisdemethylsibutramine, homosibutramine, fenfluramine, phenylpropanolamine, pentamethasone, lorcaserin, amphetamine, benzylsibutramine, chlordiazepoxide, rimonaban, sertraline, ephedrine, caffeine, theophylline, glibenclamide, glimepiride, glipizide, metformin, phenformin, pioglitazone, rosiglitazone, orlistat, furosemide, metoprazine, indapamide, bumetanide, spironolactone, and levonorgestrel. Uric acid (ethacrynic acid), eplerenone, acetazolamide, sulfanilamide, chlorothiazide, hydrochlorothiazide, chlorothiazide, methacrothiazide, epithiazide, benzylthiazide, cyclothiazide, benzylfluthiazide, cyclopenthiazine, probenecid, triamterene, phenolphthalein, bisacodyl, bisphenol salbutamol acetate, bispropofol, bisphenol salbutamol, phenolbutamol, phenolbutamol dicyclopropionate, 4-chlorophenolbutamol, 4-chlorobisisobutyrolactonebutamol, dicyclohexylphenolbutamol, deacetylated bisacodyl, fluoxetine, simvastatin, bezafibrate, benzocaine, clenbuterol, ractopamine, salbutamol.

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