| Recently,the phenomenon of illegal additions have occurred frequently with the expansion of health care market,among which the dietary supplement(DS)helps to decrease blood glucose are particularly prominent.Hypoglycemic drugs such as sulphonylureas,biguanides,meglitinides and glitazones are often added to the DS due to their good hypoglycemic action and low price.To ensure the standardization and security of market,there are many methods for rapid screening of illegal additives in DS.However,there are all with defects such as simple pretreatment,single detection methods,and time-consuming.This article studied the rules of formation of two-phase system between different short-chain alcohols and water systematically,and compared the extraction of common illegal additives by different two-phase systems in health products.Based on the combination of two-phase system and multi-detection methods,the multi-dimentional and multi-phase HPLC(high performance liquid chromatography)fingerprints were established.Fingerprint profile and similarities of chromatographic peaks could be used to identify the exogenous substances in DS,and then the results were further confirmed by multi-dimensional chromatographic fingerprints.In this study,11 common illegal additives were selected as subjects and the results were as follows.1.10 batches of bitter melon and ginseng soft gels of different brands were extracted with methanol,and illegal additives were added to the most complicated samples for separation.Four sets of fingerprints were established for the screening of 11 additives by different separation and detection methods.Accordingly,the linearities were good(R~2>0.99),and the limits of detection of the targets were 0.115~2.72 mg/kg while the recoveries were in the range of 78.07%to 99.58%,which RSD were between 1.2%~3.2%.This method was applied to the determination of 16batches of samples on the market and found that three of them contained tolbutamide,glimepiride and metformin,respectively,which were identificated by LC-MS(liquid chromatography-mass spectrometry).2.In order to expand the application of fingerprints,9 batches of DS(with different types)were selected for analysis,and 11 additives were added into the most complex samples,extracted with two-phase system(butanol and water).Biguanides were extracted into the aqueous phase while the remaining nine additives were extracted into the organic phase,and the chromatographic fingerprints(multi-phase fingerprints)were established.Targets with multiple characteristic absorption wavelengths could be detected for two times,while others with only one wavelength could be derivatized,respectively.The results showed that the linearities were good(R~2>0.99)and the limits of detection of the targets in both phases were 6.00 to 45.0 mg/kg and 17.0 to 25.0 mg/kg,respectively.4positive samples were successfully screened out by this method in 15 DSs,and the recoveries were in the range of 78.93%to 98.61%(RSD?3.2%).3.Due to the large difference of the polarity of illegal additives,two biguanide peaks were separated by using the multi heart-cutting mode of two-dimensional liquid chromatographic.Then 11 kinds of illegal additives could be analyzed at the same time,and the multi-dimensional chromatographic fingerprints were established by combining the spectral characteristics of the targets.The results showed that the limits of detection of all antidiabetics were between 5.00 and 22.0 mg/kg,and the linearities were good(R~2>0.99).The positive samples were added with three different concentrations of illegal additives,the recoveries were78.55%~95.58%,and the RSDs were 0.47%~3.2%.This method was used to analyze the above samples and the results were the same.4.10 batches of bitter melon and ginseng soft gels were studied by UPLC-Q-Orbitrap MS(ultra performance liquid chromatography coupled with quadrupole orbitrap tandem mass spectronetry).Accordingly,the multi-dimensional fingerprints of LC-MS/MS were established by Data-Dependent Full Scan MS/MS,which allows for the rapid high-throughput screening of antidiabetics in samples.Extracts were identificated by fingerprints of TIC(total ion chromatogram)firstly,and then simplify sample information of complicated matrixs by MEICs(mixed extract ion chromatograms).Finally,the positive samples were confirmed by products ion of corresponding standards.The results showed higher sensitivity,less time,and good linearity(R~2>0.99).The limits of detection of 11 antidiabetics were all between 4.60 and 27.0ng/kg and the recoveries were in the range of 70.92%to 115.0%(RSD?7.9%).This method was successfully applied to the analysis of 16 samples,and 3 of them were positive.5.Different kinds of samples were chose for the analysis,and the multi-dimensional fingerprint werealso established by the Data-Dependent Full Scan MS/MS.To reduce the matrix effect of LC-MS/MS determination of antidiabetics in DSs,mass spectrometry parameters were optimized,such as the flow rate of sheath gas and aux gas.The results showed higher ionization efficiencies and good linearities(R~2>0.99).The limits of detection of the simplest and the most complicated samples were 2.10~11.0 ng/kg and 2.30~19.0 ng/kg,while the accurancies of each was 72.42%~113.6%and 66.85%~111.0%,respectively.2 positive samples were screened out successfully by the multi-dimensional fingerprints of LC-MS/MS in the 20 samples.6.For the matrix effect of LC-MS/MS in the determination of illegal additives in hypoglycemic dietary supplement,the factors including target concentration,the number of charged ions,the chemical structure and the matrix chemical environment were studied based on the ionization efficiency and ratio of ion abundance of targets.Meanwhile,some relevant mechanisms were discussed.The results indicated that the ion abundances of targets increased linearly until it exceeded 400 ng/mL;the ion abundances of targets decreased when the number of ions in the C-trap exceeded 1×10~6;in the positive ion mode,the ionization reactions occurred easily with more electron-withdrawing groups contained in the chemical structures of targets,or otherwise;the ionization efficiencies of targets gradually decreased when the complexity of the matrix increased,that is the matrix suppression effect,while the ionization efficiencies of some targets increased,which named matrix enhanced effects. |
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