【作者】 张毅；

【导师】 崔晞；

【作者基本信息】 山东大学 ， 卫生检验学， 2015， 硕士

【摘要】 [目的]随着老龄化社会的到来,糖尿病患者数量逐渐增多,人们对自身健康状况关注度不断增加。近年来人们生活水平的提高,促进了保健品市场的繁荣,但是对于市售保健品的功效和安全性的负面报道层出不穷。因此对降糖保健品开展质量监测工作,在保障糖尿病患者的合法权益、提高患者生活质量方面有重要意义。目前缺乏对市售降糖保健品进行全面的市场调查的数据,也很少有针对济南市售降糖保健品的质量及是否添加西药成分的检测的报道。本研究拟在建立一种高效、灵敏、快速的高效液相色谱-二极管阵列检测法(HPLC-DAD),司时检测样品中降糖西药二甲双胍、格列本脲、格列吡嗪及格列美脲,通过在济南市区各大药店、超市及保健品市场进行随机抽样采集降糖保健品,对其产品质量及非法添加的降糖西药进行检测,并对样品进行功效成分,污染物限量指标铅(Pb)、砷(As)、汞(Hg),微生物指标菌落总数、大肠菌群、霉菌与酵母菌的检测,依据国家标准对降糖保健食品进行质量评价,对不合格原因进行分析,为国家监管与人们对保健品的消费提供依据。[方法]1样品采集1.1采集方法在对济南保健品市场考察的基础上,确定采样地点后,采用完全随机抽样方法,从各大超市、药店、保健品市场采集降糖保健食品。1.2样品数量共采集17种27批降糖保健品,在超市及连锁药店采集8种15批次,在保健品市场采集9种12批次。采样结束后,对样品进行编号,分类整理。2西药成分添加的检测2.1样品前处理胶囊类样品,取胶囊内容物混匀；粉末状样品,直接混匀；片剂、袋泡茶、饼干或者麦片,先用研钵研碎混匀；待测样品都细碎混匀后,再精密称取一定质量,置容量瓶中,用甲醇超声提取后,定容至刻度。胶囊壳溶液的制备,任取胶囊颗粒若干,倾出胶囊内容物,用棉花擦拭干净胶囊壳,剪成碎片,精密称取剪碎的胶囊壳,置于容量瓶中,加入50%甲醇水,60℃水浴充分溶解后,冷却至室温,用50%甲醇水定容,活性炭振摇,除去色素,过滤。2.2色谱条件色谱柱：C18(250 mm×4.6 mm,4.6μm,大连依利特)；流动相A为甲醇,流动相B为0.02 mo1/L的磷酸二氢铵(十二烷基硫酸钠0.06%,三乙胺与磷酸调pH至3.0±0.05)；流速：1.0 mL/min:柱温：25℃；进样量：20 μL；检测波长：230nm。梯度洗脱程序：0～-6 min,A:B=60:40(V:V);6～8min,A:B:60～70:40～30 (V:V)；8～16 min,A:B=70:30(V:V):16～18min,A:B=70～60:30～40(V:V)； 18～25 min,A:B=60:40(V:V)3功效成分的检测3.1检测项目从样品包装上确定待检测的功效成分有以下11种,总皂甙、总黄酮、总多糖、粗多糖、葛根素、膳食纤维、苦瓜甙、毗啶甲酸铬、三价铬(Cr3+)、铬(Cr)、锌(Zn)。其中只含有一种功效成分的样品有9种、12批次,含有两种以上功效成分的样品有8种、15批次。将功效成分分为以下几组进行检测：总皂甙,总黄酮,总多糖和粗多糖,葛根素,膳食纤维,苦瓜甙,吡啶甲酸铬,Cr3+,Cr. Zn.3.2检测方法3.2.1总皂甙依据检测标准,卫生部《保健食品检验与评价技术规范(2003版)》保健食品中总皂甙的测定,样品处理后,用紫外-可见分光光度法进行检测。3.2.2总黄酮依据检测标准,卫生部《保健食品检验与评价技术规范(2003版)》保健食品中总黄酮的测定,样品处理后,用紫外-可见分光光度法进行检测3.2.3总多糖和粗多糖依据《保健食品功效成分检测方法(白鸿主编)》粗多糖的苯酚硫酸分光光度测定法检测粗多糖,3.2.4葛根素依据检测标准GB/T 22251-2008《保健食品中葛根素的测定》,样品处理后,采用HPLC法进行检测。3.2.5膳食纤维依据GBT 5009.88-2008《食品中膳食纤维的测定》进行检测。3.2.6苦瓜甙样品处理后,采用HPLC法进行检测β-固甾醇的含量,由此计算保健食品中苦瓜甙的含量。3.2.7吡啶甲酸铬依据检测标准,卫生部《保健食品检验与评价技术规范(2003版)》保健食品中吡啶甲酸铬的测定,样品处理后,采用HPLC法进行检测3.2.8 Cr3+样品处理后,用原子吸收分光光度法进行检测,分别检测总Cr及Cr6+,二者含量之差即为Cr3+的含量。3.2.9 Cr、Zn依据GB/T 5009.123-2003《食品中铬的测定》、GB/T 5009.14-2003《食品中Zn的测定》,样品微波消解处理后,分别用原子吸收分光光度法进行检测Cr、Zn的测定。4污染物限量指标Pb、As、Hg的测定分别依据GB 500912-2010《食品安全国家标准食品中铅的测定》、GB/T5009.11-2003《食品中总砷及无机砷的测定》、GB/T 5009.17-2003《食品中总汞及有机汞的测定》,样品用微波消解法处理,定容后,用石墨炉原子吸收法测定Pb,用原子荧光法测定As、Hg5微生物指标的测定分别按照国标方法GB 47892-2010 《食品安全国家标准食品微生物学检验菌落总数测定》、GB 4789.3-2010《食品安全国家标准食品微生物学检验大肠菌群计数》、GB 478915-2010《食品安全国家标准食品微生物学检验霉菌和酵母计数》进行菌落总数、大肠菌群、霉菌和酵母菌的测定。[结果]1总体检测情况本次调查检测四大类项目,某批次保健品在任何一项检测的结果不合格即认为该产品不合格。本次采集的27批样品中,共有23批样品不合格,不合格率为85%。2降糖保健品中可能添加的西药成分检测结果2.1降糖保健食品中西药成分添加的方法学研究在给定条件下,二甲双胍、格列吡嗪、格列本脲及格列美脲得到了较好的分离,峰形良好。保留时间依次是二甲双胍(4.98min)、格列吡嗪(8.00min)、格列本脲(15.40 min)、格列美脲(18.04 min)。四种降糖西药在4～80 mg/L的浓度范围内,线性关系良好,相关系数r均在0.999以上。二甲双胍：y=29931.84x—37758.17(r=0.9995),加标回收率在96.12%～102.7%范围内,日内精密度RSD日内在2.08%～4.24%范围内,日间精密度RSD日间在3.26%-5.66%范围内,检出限是0.025 mmg/L；格列吡嗪：y=18359.70x—26693.43(r=0.9994),加标回收率在97.09%～101.8%范围内,日内精密度RSD日内在2.28%～4.27%范围内,日间精密度RSD日间在4.08%～5.21%范围内,检出限是0.044 mg/L。格列本脲：y=19189.23x+6878.51(r=0.9993),加标回收率97.86%～101.4%范围内,日内精密度RSD日内在2.27%-4.62%范围内,日间精密度RSD日间在3.29%-5.10%范围内,检出限是0.039 mg/L。格列美脲：y=17946.35x-9422.86(r=0.9993),加标回收率在97.23%-101.6%范围内,日内精密度RSD日内在2.43%4.77%范围内,日间精密度RSD日间在3.08%-5.21%范围内,检出限是0.035 mg/L。2.2降糖保健品中可能添加的西药成分及含量测定结果27批样品中,阳性样品的检出率为22.2%,有6批样品可能添加了降糖西药,其中有2批样品的胶囊壳内可能添加二甲双胍,未有样品添加格列美脲。二甲双胍的检出率为7.4%,阳性样品含量分别为7.01 mg/g、1.69 mg/g,每人每日摄入量分别是7.37 mg/d、1.01 mg/d；格列吡嗪的检出率为7.4%,阳性样品含量分别为0.46 mg/g、0.47 mg/g,每人每日摄入量分别是0.83 mg/d、6.41 mg/d；格列本脲的检出率为7.4%,阳性样品含量分别为13.73 mg/g、2.13 mg/g,每人每日摄入量分别是15.44 mg/d、4.27 mg/d。3降糖保健品中功效成分含量测定结果27批样品中,功效成分不合格样品的检出率为63.0%,功效成分不合格样品的检测值占企标最低有效剂量的比例在14.5%～96%范围内。4降糖保健品中重金属污染限量指标测定结果27批样品中,按照GB 16740-1997进行评价,重金属含量超标率为22.2%,其中Pb超标率为18.5%,As超标率为3.7%,Hg超标率为3.7%；按照GB16740-2014进行评价,重金属含量超标率为11.1%,其中Pb超标率为7.4%,As超标率为3.7%,Hg超标率为3.7%。Pb的每天摄入量占每人每天耐受摄入量(PTDI)的比例在0.01%～64.5%范围内,As的每天摄入量占每人每天最高允许摄入量(PTDI)的比例在0.00004%～0.675%范围内,Hg的每天摄入量占每人每天耐受摄入量(PTDI)的比例在0.01%～5.03%范围内。5降糖保健品中微生物指标测定结果27批样品中,微生物指标不合格样品的检出率为3.7%,未有霉菌与酵母菌超标样品;菌落总数超标样品的检出率为3.7%,超标样品的菌落总数为5125cfu/g；大肠菌群超标样品的检出率为3.7%,超标样品的大肠菌群为27 MPN/g。[结论]1济南市售降糖保健品质量较差,合格率较低。2建立的同时检测二甲双胍、格列吡嗪、格列本脲、格列美脲的HPLC-DAD法,高效、灵敏、快速,适合批量检测。3济南市售降糖保健品的西药成分添加的情况较严重。4功效成分含量合格率较低是影响济南市售降糖保健品合格率的最主要的原因。5Pb、Hg、As超标的现象较严重,其中Pb超标最严重。6济南市售降糖保健品微生物超标的现象不严重,未有霉菌和酵母菌超标的样品。更多还原

【Abstract】 [Objectives]With the advent of an aging society, the number of patients with chronic diseases such as diabetes gradually increases, and people are more and more concerned about their own health. The improvement of people’s consumption level, has promoted the consumption of dietary supplements and the prosperity of the dietary supplements industry, but it is unknown whether dietary supplements are safe and effective, therefore, the supervision of hypoglycemic dietary supplements is of great importance to diabetic patients in keeping them healthy and improving the quality of their lives. Currently, there are few survey reports on quality levels and detection of western medicines adulterated in dietary supplements in Ji’nan. In our research, the hypoglycemic dietary supplements from major pharmacies, supermarkets and health food markets were collected to explore whether the western medicines such as metformin, glipizide, glibenclamide and glimepiride, were illegally added by detection using high-performance liquid chromatography-diode array detector (HPLC-DAD) which is efficient, sensitive and rapid. At the same time, the functional compositions, heavy metal pollutants including lead (Hg)、arsenic (As)、mercury (Hg), and microbial indicators including colonies, coliform bacteria, fungi and yeast were also detected based on national standards. Our study was aimed to provide essential data for government to supervise the health food markets as well as advice for consumers.[Methods]1 Sample collection1.1 collection methodHypoglycemic dietary supplements were randomly collected from major pharmacies, supermarkets and health food markets in jinan based on the result of early investigation and arrangement.1.2 Number of samples17 kinds and 27 batches were collected altogether.8 kinds and 15 batches were from major pharmacies and supermarkets, and 9 kinds and 12 batches were from health food markets. The samples were numbered and sorted after collection.2 Detection of anti-diabetic medicine added in dietary supplements2.1 Sample pre-treatmentThe capsule samples were taken from several capsules and mixed, and the samples such as tablet, tea, biscuit or cereal were firstly crushed with a pestle and mixed later. The mixed samples were accurately weighed, put in a volumetric flask, ultrasonic assistant extracted with methanol, and diluted with methanol to volume.The capsule shell samples were made by taking a number of capsules, pouring out the contents of the capsules, wiping the capsule shells clean with cotton, then cutting them into pieces, accurately weighing, placing them in a volumetric flask, and adding 50% methanol aqueous solution, The samples were digested in a water bath in constant temperature(60℃) till the capsule shells were fully dissolved, cooled to room temperature, diluted with 50% methanol aqueous solution to volume, and then activated carbon was added to remove pigments.2.2 Chromatographic conditionsA Dalian Elite Hypersil ODS C18 column (250 mm×4.6 mm,4.6 μm) was used for the separation of four western medicines. The gradient elution was applied using methanol (A) and 0.02mol/l ammonium dihydrogen phosphate (B) containing 0.06% sodium dodecyl sulfate as well as pH adjusted 3.0 using triethylamine and phosphoric acid) and programmed as follows:the gradient started with 60% A and 40% B for 6 min, then changed to 70% A and 30% B over the next 2 min, and the compositon was held over the next 8 min then returned to 60% A and 40% in the next 2 min,and was retained in the final 7 min. The flow rate was 1.0 ml/min and the column temperature was maintained at 25℃. The injection volume was 20 μl and 230 nm was chosen for routine monitoring.3 Detection of functional compositions3.1 Detection itemsThe items determined from the sample package were the following 11 ingredients, total saponins, total flavonoids, total polysaccharides, coarse polysaccharides, puerarin, dietary fiber, bitter glycosides, chromium picolinate, trivalent chromium (Cr3+), chromium (Cr), zinc (Zn).9 kinds and 12 batches of samples contain only one kind of functional composition, while 8 kinds and 15 batches contain more than two kinds of functional compositions. The items detected were divided into the following groups, total saponins, total flavonoids, total polysaccharides and coarse polysaccharides, puerarin, dietary fiber, bitter glycosides, chromium picolinate, Cr3+, Cr and Zn.3.2 Detection method3.2.1 Total saponinsAfter sample treatment, total ginsenosides were detected using UV-vis spectrophotometry, based on "Technical standards for testing and assessment of health food (2003 edition) Determination of total ginsenosides in health food"3.2.2 Total flavonoidsAfter sample treatment, Total flavonoids were detected using UV-vis spectrophotometry, based on "Technical standards for testing and assessment of health food (2003 edition) Determination of total flavonoids in health food"3.2.3 Total polysaccharides and coarse polysaccharidesUse phenol-sulfuric acid UV detection to test coarse polysaccharides, based on "Standards for functional ingredients of health food".3.2.4 PuerarinAfter sample preparation, use HPLC for detection, based on GB/T 22251-2008" determination of puerarin in health foods ".3.2.5 Dietary fiberBased on GBT 5009.88-2008 " Determination of dietary fiber in foods " for testing.3.2.6 Bitter glycosidesAfter sample preparation, use HPLC for β-solid sterol detection, and thus calculate the contents of bitter glycosides in health foods.3.2.7 Chromium picolinateAfter sample preparation, use HPLC for chromium picolinate detection, based on"Technical standards for testing and assessment of health food (2003 edition)"3.2.8 Cr3+The samples were processed, and Cr was detected by atomic absorption spectrophotometry. The difference between the contents of total Cr and Cr6+ is the content of Cr3+.3.2.9 Cr and ZnAfter microwave digestion treatment, use atomic absorption spectrophotometry to detect Cr and Zn, based on GB/T 5009.123-2003 " Determination of chromium in foods" and GB/T 5009.14-2003 " Determination of zinc in foods "4 Detection of heavy metal pollutants Pb, As and HgAfter microwave-digestion to digest the samples, use graphite furnace atomic absorptionspectrometry to detect Pb and atomic fluorescence spectrometry to detect As and Hg, based on GB 500912-2010 "National food safety standard Determination of lead in foods"、GB/T 5009.17-2003 "Determination of total arsenic and abio-arsenic in foods" and GB/T 5009.17-2003 "Determination of total mercury and organic-mercury in foods"5 Detection of microbial indicatorsAerobic plate count, enumeration of coliforms, and enumeration of moulds and yeasts were based on GB 47892-2010 "National food safety standard Food microbiological examination:Aerobic plate counf"、GB 4789.3-2010 "National food safety standard Food microbiological examination:Enumeration of coliforms" GB 478915-2010 "National food safety standard Food microbiological examination: Food microbiological examination:Enumeration of moulds and yeasts"[Results]1 The overall detectionFour categories of items were detected, and either one of them unqualified was named defect. In the 27 batches of samples,23 batches were unqualified, and the defect rate was 85%.2 Situation of anti-diabetic agents adulterated in dietary supplements2.1 The method study for the determination of four anti-diabetic agents (metformin, glipizide, glibenclamide and glimepiride)Under the given conditions, metformin, glipizide, glibenclamide and glimepiride were completely separated and all of them had good peak shapes. The retention times of metformin, glipizide, glibenclamide and glimepiride were 4.98min,8.00 min,15.40 min,18.04 min. In the concentration range of 4～80 mg/L, the four antidiabetic medicines had good linear relationship (r> 0.999). Metformin:y=29931.84x-37758.17 (r=0.9995); the recoveries:96.12%～102.7%; the inter-day RSD: 2.08%～4.24%; the intra-day RSD:3.26%-5.66%; the detection limits:0.025 mg/L. Glipizide:y=18359.70x-26693.43 (r=0.9994); the recoveries:97.09%-101.8%; the inter-day RSD:2.28%～4.27%; the intra-day RSD:4.08%～5.21%; the detection limits: 0.044 mg/L. Glibenclamide:y= 19189.23x+6878.51 (r=0.9993); the recoveries: 97.86%～101.4%; the inter-day RSD:2.27%-4.62%; the intra-day RSD: 3.29%-5.10%; the detection limits:0.039 mg/l.Glimepiride:y= 17946.35x-9422.86 (r=0.9993), the recoveries:97.23%～101.6%; the inter-day RSD:2.43%-4.77%; the intra-day RSD:3.08%～5.21%; the detection limits:0.035 mg/L.2.2 The kinds and contents of anti-diabetic agents adulterated in dietary supplementsIn the 27 batches of samples, the detection rate of positive samples was 22.2%, in which 6 batches of samples might add anti-diabetic agents, and 2 batches of capsule shells might add metformin, while no sample was glimepiride-adulterated. The results were as follows:the detection rate of metformin was 7.4%; the contents in the positive samples were 7.01mg/g and 1.69mg/g; the contents of daily intake were 7.37 mg/d and 1.01 mg/d; the detection rate of glipizide was 7.4%; the contents in the positive samples were 0.46 mg/g and 0.47 mg/g; the contents of daily intake were 0.83 mg/d and 6.41 mg/d; the detection rate of glibenclamide was 7.4%; the contents in the positive samples were 13.73 mg/g and 2.13 mg/g; the contents of daily intake were 15.44 mg/d and 4.27 mg/d.3 Situation of functional compositions detectionIn the 27 batches of samples, the defect rate was 63.0%, and the percentage of contents of unqualified samples to the lowest effective dose of the standard was in the range of 14.5% to 96%.4 Situation of heavy metal pollutants detectionOf the 27 batches samples, when evaluated in accordance with GB 16740-1997, the total unqualified rate was 22.2%, the unqualified rate of Pb was 18.5%, the unqualified rate of As was 3.7%, and the unqualified rate of Hg was 3.7%. When evaluated in accordance with GB 16740-2014, the total unqualified rate was 11.1%, the unqualified rate of Pb was 7.4%, the unqualified rate of As was 3.7%, and the unqualified rate of Hg was 3.7%.The percentage of accounts of Pb daily intake to provisional tolerable daily intake (PTDI) was in the range of 0.01%～64.5%. The percentage of accounts of As daily intake to PTDMI was in the range of 0.00004%～0.675%. The percentage of accounts of Hg daily intake to PTDI was in the range of 0.01%～5.03%.5 Situation of microbial indicators detectionIn the 27 batches of samples, the unqualified rate was 3.7%, while no excessive enumeration of moulds and yeasts samples existed; The unqualified rate of the total colony count was 3.7%, and total aerobic plate count which had exceed the standard was 5125 cfu/g; The unqualified rate of enumeration of coliforms was 3.7%. and enumeration of coliforms which had exceed the standard was 27 MPN/g.[Conclusions]1 Hypoglycemic dietary supplement in jinan had bad quality and low qualified rate.2 The established method, HPLC-DAD, for detection of metformin, glipizide, glibenclamide and glimepiride was efficient, sensitive, rapid, suitable for batch testing.3 Situation of antidiabetic agents added in dietary supplements in jinan was seriously bad.4 The main reason for low total qualified rate was that the contents of functional compositions were unqualified.5 Situation of Pb, Hg, As pollution was serious, and the most serious was Pb pollution.6 Situation of microbial indicators which had exceed the standard was not serious, and no sample which had excessive mold and yeast.更多还原