| Sulphur fumigation, mainly used in traditional Chinese medicine (TCM) that is difficult to dry or easy to rotten, is a traditional processing method in producing areas. The sulphur burning generates sulfur dioxide, which has the function of sterilization, insect prevention and mould proof, thus benefit for storage and maintenance of TCM. However, the appearance, chemical compositions and pharmacological effects of TCM may be changed after sulphur fumigated, and the main residual sulfites have toxic effects on the body’s nervous system, digestive system and respiratory system. In2004, SFDA provided that sulphur fumigated TCM in market were substandard medicines. Chinese pharmacopoeia2005edition volume I had already deleted the sulphur fumigation technology of yam and kudzu root. In June2011, China Pharmacopoeia Committee published the limits of sulfur dioxide residues in TCM, said that the sulfur dioxide residues in eleven kinds of TCM were not more400μg·g-1and others less than150μg·g-1. Chinese pharmacopoeia2010edition volume1had set the sulfur dioxide residues determination method in appendix IX U, acidized the samples in airtight containers to distill sulfur dioxide and titrated by Iodine titrant, thus calculated the sulfur dioxide residues in TCM. However, the detection devices were complex, the operation process was cumbersome and the Iodine titrant should be calibrated by Arsenic trioxide before use. Therefore, the official method is not conductive to promote.Based on the phenomenon that sodium sulfite reacted with OPA and ammonium acetate to generate purple color materials, this paper established a method to analyze the sulfur dioxide residues in TCM qualitatively and quantitatively. Based on the principle that sulfites reacted with OPA and ammonium salt to generate fluorescent chemicals, this paper first established a derivative fluorometry method for the determination of sulfur dioxide residues in TCM. Based on the principle that reversible combinated sulfites in TCM can be released by alkaline solution, this paper first established the method of shaking extraction with alkaline solution to use in sample pretreatment. The established method was used to determine the sulfur dioxide residues of yam, kudzu root, astragalus, codonopsis pilosula, angelica, chrysanthemum and semen coicis. This method is simple for operation, quick and sensitive. It can provide reference for the quality control of sulphur fumigated TCM. The main contents and results were as follows:1. Study on the rapid detection method of sulfur dioxide residues in TCM Based on the phenomenon that sodium sulfite reacted with OPA and ammonium acetate to generate purple color materials, and the color chromaticity had positive relationship with the addition of sodium sulfite, this paper established a method to analyze the sulfur dioxide residues in TCM qualitatively and quantitatively. A linear relationship was obtained between the color chromaticity and the addition of sodium sulfite in the range of0～25.23μg. This method was used to detect the sulfur dioxide residues in a total of20batches purchased samples, the results showed that the detected samples all had sulfur dioxide residues. Compare the quantitative results with the limit standard of sulfur dioxide residues in TCM, the sulfur dioxide residues in all the samples of yam, kudzu root, astragalus and in part of the samples of codonopsis pilosula and semen coicis were more than the limits, showed that sulphur fumigated TCM was widespread. This method is simple for operation, quick and sensitive. It can provide a reference for the fast detection of sulfur dioxide residues in TCM.2. Study on the determine method of sulfur dioxide residues in TCM2.1Study on the establishment of derivative fluorometry method2.1.1Establishment of best fluorescenet derivative conditionsSulfur dioxide residues in TCM mainly exist in the form of dissociative state and combine state. The dissociative state include HSO3-、SO2and SO32-, and can easily react with reducing sugar, protein, pigment, enzyme, vitamin, aldehyde and ketone in TCM to form combine state. Because the irreversible combined sulfites don’t dissociate in physiological environment and won’t damage human health. Therefore, the detect targets of sulfur dioxide or sulfites are dissociative SO32-and reversible combined sulfites.Chinese pharmacopoeia2010edition volume I had set the sulfur dioxide residues determination method in appendix IX U, acidized the samples in airtight containers to distill sulfur dioxide and titrated by Iodine titrant, thus calculated the sulfur dioxide residues in TCM. However, the detection devices were complex, the operation process was cumbersome and the Iodine titrant should be calibrated by Arsenic trioxide before use. Therefore, the official method is not conductive to promote. The methods of determining sulfur dioxide residues in TCM reported in literature mainly include hydrochloric para-roanilin method, IC, HPLC and GC. The hydrochloric para-roanilin method need to use sodium tetrachloromercurate absorption which is toxic, while the chromatography will increase detecting costs. Based on the principle that sulfites reacted with OPA and ammonium salt to generate fluorescent chemicals, this paper first established a derivative fluorometry method for the determination of sulfur dioxide residues in TCM. The best condition of fluorescent detivative was achieved by single factor investigated on pH, dosage of derivative reagents, reaction temperature and time. This method is simple for operation with quick and correct result and sensitive. It can provide a reference for the determination of sulfur dioxide residues in TCM.2.1.2Study on the sample pretreament methodChinese pharmacopoeia2010edition volume Ⅰ had set the pretreament method in appendix Ⅸ U. However, the detection devices are complex, the operation process is cumbersome. The reversible combined sulfites are stalbe in acidic conditions, and separate into SO32-when pH>7.This paper studyed on the sample pretreament method. Base on the principle that the reversible combined sulfites in TCM could be released by alkaline solution, this paper took yam as research object. First, Plackett-Burman Design was used to screen out the significant influencing factors in the pretreatment, then Central Composite Design-response Surface Methodology was used to optimize the factors. This paper first established the method of shaking extraction with alkaline solution to use in sample pretreatment and can provide reference for sample pretreatment in the determination of sulfur dioxide residues in TCM.2.2Determining of sulfur dioxide residues in TCM2.2.1Study on the validation of the established fluorescent derivative methodThis paper took yam without sulphur fumigated as research object, sulphur fumigated for30,60,90,120and150min, extracted these samples with alkaline solution by shaking extraction method. then determined the sulfur dioxide residues in samples of different sulphur fumigated time by fluorescent detivative method. The results showed that the sulfur dioxide residues of samples without sulphur fumigated were0, while the sulphur fumigated samples all had sulfur dioxide residues, and the sulfur dioxide residues had a positive relationship with sulphur fumigation time. The results show that it’s feasible to determine the sulfur dioxide residues in TCM by fluorescent derivative.2.2.2Study on the method comparision of in this paper and in Chinese pharmacopoeia2010edition volume Ⅰ Pretreated the samples of yam, codonopsis pilosula, angelica, and semen coicis with alkaline solution, then determined the sulfur dioxide residues in these samples. Compared with the method in Chinese pharmacopoeia2010edition volume Ⅰ, the determination results of the method in this paper were higher, speculated that Pretreated with alkaline solution could release reversible combined sulfites, while acidic solution could only release free sulfites, so the determination results of the method in this paper were more close to the actual sulfur dioxide residues in TCM.2.2.3Sample determinationDetermined the sulfur dioxide residues of yam, kudzu root, astragalus, codonopsis pilosula, angelica, chrysanthemum and semen coicis with the established method, compared with the limit standards, some purchased samples were high than the limits. The results show that sulphur fumigation in TCM is still widespread.3Study on the draft standards formulation of sulfur dioxide residues determina-tion in TCMThis paper had done comprehensive and systematic research on the determination of sulfur dioxide residues by fluorescent derivative, thus formulated the draft standards of sulfur dioxide residues determination in TCM. The results can provide scientific basis for quality control of sulphur fumigated TCM. |
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