保健食品中褪黑素的測定 編制說明

《保健食品中褪黑素的測定》征求意見稿）編制說明一、工作簡況根據(jù)《國家標準化管理委員會關于下達2023年第二批推薦性國家標準計劃及相關標準外文版計劃的通知》（國標委發(fā)〔2023〕37二、國家標準編制原則、主要內容及其確定依據(jù)將標準名稱修訂為“保健食品中褪黑素的測定”。在適用范圍中明確褪黑素作為主要功能成分。將標準適用范圍修本文件描述了保健食品中褪黑素的液相色譜測定方法。本文件適用于以褪黑素作為主要功效成分添加于保健食品中的褪黑素的測定。4.儀器條件的優(yōu)化原有國標溫度為室溫，分別測試褪黑素標準品(5ppm、50ppm)在25℃、30℃、35℃的響應，結果詳見表1,結果表明不同溫度下褪黑素的峰面積差異不大，考慮到季節(jié)變化以及實驗室環(huán)境，將柱標準溶液濃度(μg/mL)同時，驗證了高效液相色譜-熒光檢測法的測定方法，對比結果顯示方法等效。詳見圖1和圖2。經(jīng)過方法優(yōu)化，儀器條件為：色譜柱：Cl?柱(粒徑5.0μm,4.6mm×250mm),流動相(5.2.2):甲醇+三氟乙酸水(45+55,體積比);進樣體積：10μL:5.試樣前處理條件的優(yōu)化增加了試樣的劑型種類，包括片劑、粉劑、顆粒、軟膠囊和硬膠囊、口服液、凝膠糖果劑型，明確制備步驟。(2)試樣處理方式優(yōu)化①含淀粉試樣對比70%乙醇對不同含量、不同劑型的褪黑素產(chǎn)品的提取效果，根據(jù)提取率確定提取次數(shù)。首先在空白片劑(0.2g)基質中添加不同水平的褪黑素標準品。綜合四種添加水平不同提取次數(shù)對應的提取效果，確定提取次數(shù)為2次。②含油試樣三、試驗驗證的分析、綜述報告四、與國際、國外同類標準技術內容的對比情況五、以國際標準為基礎的起草情況六、與有關法律、行政法規(guī)及相關標準的關系七、重大分歧意見的處理經(jīng)過和依據(jù)八、涉及專利的有關說明九、實施國家標準的要求，以及組織措施、技術措施、過渡期和實施日期的建議建議本標準發(fā)布6個月后實施，由歸口單位組織行業(yè)相關單位十、其他應當說明的事項附件：方法學驗證報告附件1.特異性國家市場監(jiān)督管理總局制定的《輔酶Q1o等五種保健食品原料備案產(chǎn)品劑型及技術要求》中以褪黑素為保健食品原料時，可以以單一褪黑素原料備案，也可同時加入維生素B?作為原料進行產(chǎn)品備案?，F(xiàn)有部分產(chǎn)品同時加入褪黑素和維生素B?,采用PDA檢測器分別測試褪黑素標準品(5ppm)和含有褪黑素和維生素B?的實際樣品，結果見圖3~圖4。采用熒光檢測器分別測試褪黑素標準品、褪黑素和吡哆醇標準品混合溶液、褪黑素和吡哆醛標準品混合溶液、褪黑素和吡哆胺標準品混合溶液，結果見圖5~圖8,結果表明維生素B?對褪黑素的檢測無干擾。1.1紫外檢測法1.2熒光檢測法圖5褪黑素標準品(5ppb)譜圖圖7含吡哆醛(100ng/mL)和褪黑素標準品(100ng/mL)譜圖圖8含吡哆胺(100ng/mL)和褪黑素標準品(100ng/mL)譜圖2.檢出限和定量限將標準品稀釋成不同濃度進行測試，用紫外檢測器檢測時，當濃熒光檢測器檢測時當濃度為0.2ng/mL時，S/N≥3,當濃度為0.5ng/mL時，S/N≥10,表2為測試結果。紫外檢測法濃度0.05μg/mL均值4.775.165.715.604.714.345.04濃度/熒光檢測法濃度0.2ng/mL/4.994.254.794.725.624.344.78濃度0.5ng/mL/3.測定范圍作液的濃度為橫坐標(x)，褪黑素特征峰峰面積為縱坐標(y)，進行線標準工作溶液(μg/mL）15R2=0.9998R2=0.99984.正確度和重復性片劑本底本底g/100g硬膠囊本底g/100g添加量g/100g實測值g/100g率%回收率范圍%RSD%添加量g/100g實測值g/100g率%回收率范圍%RSD%/0.0250.02390.02550.02460.02560.02640.026395.598.295.5~3.80.0250.02520.02640.02600.02660.02680.0264100.6~107.22.30.128102.40.127102.099.197.40.1250.1300.1310.1310.130102.0~/0.1250.1320.1160.11797.992.494.092.4~105.84.80.2450.2370.23097.994.692.10.2530.2530.2540.2500.2500.2380.24899.995.499.492.1~99.90.2500.2360.2550.25194.394.3~102.22.9凝膠糖果軟膠囊本底g/100g添加量g/100g實測值g/100g率%回收率范圍%RSD%本底g/100g添加量g/100g實測值g/100g率%回收率范圍%RSD%/0.0250.0255102.00.0258103.10.024397.20.0255102.10.0273109.0/0.0250.0250100.10.0267106.70.0265105.90.0253101.3...0.0261104.20.0261104.23369753369750.12597.90.12398.20.13197.5~.0.1290.1300089250089250.1250.11995.20.1300.1290.11692.8~.0.11793.70.12095.80.2500.24798.80.23795.00.24296.60.23895.40.24899.40.2500.259103.80.22891.20.24296.90.23895.2...0.24497.50.24597.9空白樣品含量g/100g添加量g/100g加標樣品含量g/100g回收率R%回收率范圍%RSD%/0.0250.0260.0270.0260.0260.0250.02496.496.4~106.83.60.1250.1270.1280.1150.1260.12691.798.891.7~102.74.00.2500.2530.2580.2370.2450.2480.25394.998.199.394.9~103.43.0片劑本底本底g/100g硬膠囊本底g/100g添加量g/100g實測值g/100g率%回收率范圍%RSD%添加量g/100g實測值g/100g率%回收率范圍%RSD%0.3500.9900.98897.997.497.4~3.00.55099.796.799.997.396.7~2.794.494.92.542.4799.00.6500.70090.195.24.22.492.512.492.5699.0~2.097.599.096.497.197.296.4~100.02.973.023.053.053.093.0496.599.796.5~103.72.4粉劑本底本底g/100g本底g/100g添加量g/100g實測值g/100g率%回收率范圍%RSD%添加量g/100g實測值g/100g率%回收率范圍%RSD%0.030.08240.08270.08120.08810.087190.791.788.488.4~0.0120.03450.03580.03530.03580.036794.594.5~5.00.05520.08770.02320.03570.060.1100.1170.1190.1160.11694.391.991.9~5.40.0240.04860.04900.04930.04920.04930.0490105.9~0.090.1380.1370.1370.14394.892.490.597.090.597.490.5~97.43.30.0360.06050.06200.06180.06200.06170.058598.13.7軟膠囊本底本底g/100g凝膠糖果本底g/100g添加量g/100g實測值g/100g率%回收率范圍%RSD%添加量g/100g實測值g/100g率%回收率范圍%RSD%0.5200.2750.8180.8260.7820.8140.7940.78695.399.596.895.3~4.40.06250.1500.1460.1530.1490.15490.297.590.2~5.898.20.1680.17793.90.55091.998.798.691.9~4.20.08550.08750.1800.1660.1780.17492.592.5~6.095.497.00.2140.2120.20897.70.82597.495.498.995.4~100.22.00.1250.2180.2220.20696.596.5~106.64.0回收率良好，符合GB5009.295-2023中不同濃度對方法正確度的要片劑凝膠糖果本底添加實測回收率范RSD本底添加實測回收率范RSDg/100g量g/100g值g/100g率%圍%%g/100g量g/100g值g/100g率%圍%%0.0250.02200.02420.02350.02350.02470.024287.896.893.894.098.796.987.8~98.70.02570.02630.02430.02680.02560.027397.14.1/0.1250.1200.1250.1250.1300.12797.196.299.92.8/0.1250.1200.1200.1260.1200.12795.996.099.12.795.9~0.22590.10.23292.60.22389.10.23292.80.21586.20.23895.00250862~950350250.0.23795.0...0.22891.20.250100.00.22590.10.251100.592.6~103.74.7本底本底g/100g硬膠囊本底g/100g添加量g/100g實測值g/100g率%回收率范圍%RSD%添加量g/100g實測值g/100g率%回收率范圍%RSD%0.0120.03220.03380.03400.03420.034585.098.285.0~7.30.5502.012.062.062.3298.798.4~112.75.30.02200.03452.0198.40.0240.04670.04660.04720.04660.04730.0470102.3~2.672.682.682.752.712.80108.9~2.30.0360.05780.05930.05890.05840.05830.055999.494.294.2~103.63.33.313.313.323.383.374.4軟膠囊本底本底g/100g粉劑本底g/100g添加量g/100g實測值g/100g率%回收率范圍%RSD%添加量g/100g實測值g/100g率%回收率范圍%RSD%0.2750.7550.7910.7620.7620.7580.76497.898.897.8~4.60.030.08300.08240.08220.08550.08750.083098.397.897.8~115.26.80.97087.395.30.1070.10790.689.37.40.4860.5500.9686.395.696.486.3~5.90.05290.060.1160.11796.397.989.3~107.40.82588.090.091.892.887.792.487.7~92.82.50.090.1300.1280.1330.13079.285.483.389.475.285.175.2~89.4回收率良好，符合GB5009.295-2023中不同濃度對方法正確度的要5.不同品牌褪黑素標準物質的比較選擇了五個不同品牌（安譜，Anpel；西格瑪，Sigma-Aldrich；））褪黑素標準物質，對其DAD檢測器在222nm響應情況進行比較， 保留時間穩(wěn)定情況：對5個品牌的褪黑素溶液圖譜進行疊加比—不同品牌間標準物質的峰面積相對標準