| [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. |
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