附件2 《含氨基酸水溶肥料 游离氨基酸含量的测定-分光光度法》（编辑说明）

团体标准含氨基酸水溶肥料游离氨基酸含量的测定—分光光度法编制说明2022年10月15日团体标准《含氨基酸水溶肥料游离氨基酸含量的测定—分光光度法》编制说明1.工作简况1.1任务来源根据《广西肥料协会关于下达2022年第二批团体标准项目计划的通知》（广肥协[2022]5号）的要求，团体标准《含氨基酸水溶肥料游离氨基酸含量的测定—分光光度法》制定项目由广西肥料协会归口，项目周期为6个月，完成年限为2022年12月，标准起草单位为：广西壮族自治区分析测试研究中心、广西乐土生物科技有限公司、广西垂青生物科技有限公司、广西壮族自治区地质矿产测试研究中心、广西壮族自治区产品质量检验研究院、广西西大检测有限公司等。1.2项目概况现有国家标准推荐使用的两种游离氨基酸检测方法，所使用的仪器价格昂贵（自动分析仪每台80万元以上，液相色谱仪及配套仪器10万元以上），标准溶液价格也很贵,检测成本高，而且检测周期长，结果滞后，不能及时指导生产。现立项研究的《含氨基酸水溶肥料游离氨基酸含量的测定—分光光度法》检测成本低，检测速度快，非常适合肥料生产工艺的管理和质量控制，大大节省了企业的成本。1.3承担单位情况广西壮族自治区分析测试研究中心（以下简称“中心）为公益二类事业单位，成立于1978年，是广西科技厅直属的以分析测试技术研究、理化分析为主的省级综合分析测试研究机构，是广西质量技术监督局授权建立的“广西壮族自治区保健食品及生物产品质量监督检验站”，属于为社会公众服务、对外独立开展检验/检测业务的第三方检测研究机构。中心现有职工82人，其中高级职称18人，中级职称29人，硕士研究生17人，从事质检工作76人。中心拥有固定资产7809万元，仪器设备1402台套，其中50万元以上的大型仪器37台套，20万以上仪器92台套，如气相色谱/质谱联用仪（GC/MS）、液相色谱/质谱联用仪（LC/MS）、气相色谱/红外光谱联用仪（GC/RFX）、氨基酸分析仪、全自动遗传分析仪、高效液相色谱仪、全自动超高压液相色谱仪、等离子发射光谱仪（ICP）、等离子体质谱仪(ICP/MS)、离子色谱仪（IC）、X射线荧光光谱仪、原子荧光分光光度计、原子吸收分光光度计等。历年来中心承担多项省部级重要课题，获得科技成果27项，其中广西科技进步奖8项。中心承担各类产品质量技术标准的撰写起草工作，其中3项国家行业标准、22项广西地方标准和50项企业标准已通过有关主管部门认可发布实施。中心在生物样品有效成分分析、有益活性菌分析、保健食品功效成份分析、农药残留、兽药残留检测等领域处于广西领先水平。1.4参编单位及主要起草人工作情况广西壮族自治区分析测试研究中心负责统一样品的收集和分发，分析方法的实验研究，样品测试结果的收集和处理，标准文本、试验报告和编制说明的撰写。广西乐土生物科技有限公司、广西垂青生物科技有限公司、广西壮族自治区地质矿产测试研究中心、广西壮族自治区产品质量检验研究院、广西西大检测有限公司方法验证单位，负责对试验报告中的条件试验进行验证，并对标准文本提出修改意见。标准主要起草人以及分工如表1所示。表1标准主要起草人及分工单位姓名分工广西壮族自治区分析测试研究中心黄一帆、陈桂鸾、潘扬昌、李义芬、班雁华、邓艺萍、黄殿贵、莫达松、陆云、陆银英、刘永强、陈海燕、唐小丽、许园园、陈艳、谭燚主起草单位广西乐土生物科技有限公司陆明光、吕贵升、邓多桓、滕丽娜、陈裕新共同起草单位、方法验证广西垂青生物科技有限公司韦晓社、贾虎成、陈小莉共同起草单位、方法验证广西壮族自治区产品质量检验研究院吉日文、陈海艳、陆章权共同起草单位、方法验证广西壮族自治区地质矿产测试研究中心刘辉庭、梁鑫佳、卢一叶共同起草单位、方法验证广西西大检测有限公司周权能、裴根、李辉宁共同起草单位、方法验证1.5标准起草过程广西壮族自治区分析测试研究中心接到《含氨基酸水溶肥料游离氨基酸含量的测定—分光光度法》的制定任务后，立即组织相关工程技术人员成立了标准编制组，进行相关资料的查询与收集工作，制订工作计划和进度安排。编制组首先查阅大量材料，并根据NY/T1975—2010《水溶肥料游离氨基酸含量的测定》中自动分析仪法（仲裁法）的原理，设计出一个初步的检测方案，然后再制定出实验的每一个具体操作步骤。通过大量的实验，不断完善实验步骤，优化检测方案，然后确定出一个检测结果稳定、操作简单的实验方法，再按照确定的实验方法进行大量的比对实验，根据比对实验的统计结果确定出一个矫正系数。在此基础上，于2022年10月形成了标准的讨论稿和编制说明。本标准讨论稿完成后，在编制组及单位内部进行了多次交流，广泛征求意见，对本标准讨论稿进行了认真的修改和完善，最后形成了该标准的征求意见稿。标准的制订原则、主要内容与论据2.1标准编制的原则该标准按照GB/T1.1—2020《标准化工作导则第1部分：标准化文件的结构和起草规则》的规定和要求编写。本文件坚持以生产实际的可操作性为前提，以满足其实践性、适应性、便捷性等需要为原则。2.2确定标准的主要内容2.2.1标准题目与用途本文件的立项名称为：“含氨基酸水溶肥料游离氨基酸含量的测定—分光光度法”，在标准征求意见的过程中未提出其他建议，确定为此项标准的名称。本标准规定了水溶肥中游离氨基酸含量测定的分光光度法的试验方法。本标准适用于液体或固体水溶肥料中游离氨基酸含量的测定。2.2.2试剂表1所需试剂名称规格所需浓度亮氨酸99%240mg/L茚三酮分析纯3%异丙醇分析纯10%抗坏血酸分析纯0.8g/L盐酸分析纯0.06mol/L乙二胺四乙酸二钠分析纯10g/L冰乙酸分析纯/乙酸钠分析纯/活性炭分析纯/2.2.3设备分光光度计、分析天平2.2.4试验步骤2.2.4.1试样的制备固体样品经多次缩分后，取出约100g，将其迅速研磨至全部通过0.50mm孔径筛（如样品潮湿，可通过1.00mm筛子），混合均匀，置于洁净、干燥容器中。液体样品经多次摇动后，迅速取出约200mL，置于洁净、干燥的容器中。2.2.4.2试样溶液的制备称取试样1g～5g（精确到0.001g），置于250mL容量瓶中，用水充分溶解后定容到刻度，摇匀后干过滤，弃去20mL初滤液。取约30mL滤液于小烧杯中，加入一勺（约2g）活性炭搅拌2min，静置5min后干过滤，再用0.45微米的微孔滤膜过滤滤液。准确移取0.50mL滤液于50mL容量瓶中，加入1.0mLEDTA-Na溶液（3.2.3）和1.0mL盐酸溶液（3.2.1），用酸-乙酸钠缓冲溶液定容，摇匀后干过滤，滤液备用待测。2.2.4.3标准曲线的绘制空白溶液的制备：用移液管依次吸取2.00mL蒸馏水、1.00mL茚三酮溶液、0.50mL抗坏血酸溶液、1.50mL乙酸-乙酸钠缓冲溶液于具塞比色管中，在100℃的沸水中加热15min，取出冷却至室温，摇匀。标准曲线的绘制：用移液管吸取1.00mL、2.00mL、3.00mL、4.00mL氨基酸标准溶液分别置于4个50mL容量瓶中，用10%异丙醇溶液定容，摇匀。分别用移液管依次吸取2.00mL上述溶液、1.00mL茚三酮溶液、0.50mL抗坏血酸溶、1.50mL乙酸-乙酸钠缓冲溶液于具塞比色管中，在100℃的沸水中加热15min，取出冷却至室温，摇匀。以空白溶液为参比，于波长573nm出测定各吸光度。以氨基酸标准溶液的浓度为纵坐标，吸光度为横坐标绘制标准曲线。2.2.4.4样品的测定分别用移液管依次吸取2.00mL试样溶液（3.4.2）、1.00mL茚三酮溶液、0.50mL抗坏血酸溶液、1.50mL乙酸-乙酸钠缓冲溶液于具塞比色管中，在100℃的沸水中加热15min，取出冷却至室温，摇匀。以空白溶液为参比，于波长573nm处测定样品溶液吸光度，在标准曲线上查得相应的游离氨基酸质量。2.3主要试验验证情况（1）为验证本文件测试方法的适用性，由广西壮族自治区分析测试研究中心提供代表性的含氨基酸水溶肥料样品，统一分配样品并安排验证试验。除了使用分光光度法进行测试以外，广西壮族自治区分析测试研究中心还使用国标法—高效液相色谱法进行测试。各验证实验室将分光光度法的值与高效液相法的值相除即可得到矫正系数f，然后使用统计学原理—格拉布斯检验法对f进行评价，评判最小值或最大值是否离群。表2验证试验数据样品编号系数f平均值标准偏差相对标准偏差%最小值最大值GminGmax实验室1实验室2实验室3实验室42207012.082.061.961.851.990.115.31.852.081.290.872207022.011.982.042.002.010.021.21.982.041.121.262207031.951.922.011.961.960.042.01.922.011.141.282207041.851.821.771.921.840.063.51.771.921.161.242207051.571.611.511.601.570.053.01.511.611.410.762207061.531.571.441.501.510.053.61.441.571.241.152207071.761.811.691.611.720.095.11.611.811.221.062207081.481.421.451.511.460.042.81.421.511.111.232207091.621.581.671.591.620.042.51.581.670.921.332207101.771.811.791.851.810.031.81.771.850.961.312207111.531.511.441.351.460.085.71.351.531.350.842207121.931.841.891.781.860.063.51.781.931.241.082207131.851.871.941.921.890.042.21.851.941.151.062207141.811.851.771.671.780.084.31.671.851.370.982207152.082.082.042.162.090.052.32.042.160.921.422207162.031.981.901.961.960.052.81.902.031.251.152207171.401.481.441.561.470.074.51.401.561.071.302207181.441.391.501.501.460.053.81.391.501.290.792207192.292.202.182.452.280.125.52.182.450.841.372207202.122.002.001.882.000.104.81.882.121.221.222207211.411.451.511.591.490.085.21.411.591.011.292207221.381.421.491.341.410.064.51.341.491.051.282207231.941.881.821.741.850.094.61.741.941.221.092207241.811.931.981.851.890.073.91.811.981.071.1422072512.7512.1912.6312.0012.390.352.912.0012.751.101.012207262.632.542.792.712.670.114.02.542.791.161.162207272.071.971.901.941.970.073.81.902.071.001.362207282.152.212.062.012.110.094.12.012.211.101.152207292.202.132.002.102.110.084.02.002.201.311.102207301.942.031.861.771.900.115.91.772.031.191.162207312.091.962.062.262.090.126.01.962.261.071.332207322.252.152.061.972.100.125.71.972.251.141.182207332.312.292.292.252.280.021.12.252.311.450.842207341.601.571.521.461.540.064.01.461.601.271.052207351.401.441.441.481.440.032.21.401.481.171.28220400253.763.643.713.953.760.133.53.643.950.941.39220401924.114.023.953.843.980.112.93.844.111.221541.521.631.541.560.053.31.521.630.761.47220403603.433.563.503.313.450.113.13.313.561.301.04220407281.801.851.751.641.760.094.91.641.851.351.00220500571.571.551.481.431.510.064.11.431.571.200.99220502084.724.824.714.654.730.071.44.654.821.061.35220509451.641.561.521.701.610.084.91.521.701.051403.503.563.503.490.071.93.403.561.371.03220514811.691.631.571.661.640.053.11.571.691.251.11220603473.483.573.463.863.590.185.13.463.860.731.45220500231.381.461.421.561.460.085.51.381.560.931.38220500241.691.731.901.891.800.115.91.691.901.010.90220500251.511.531.401.651.520.106.61.401.651.181.26220500261.591.521.461.521.520.063.61.461.591.191.26220501932.001.981.901.821.920.084.41.822.001.280.94220501941.841.911.931.951.900.052.51.841.951.410.89220501951.951.921.861.851.890.052.61.851.950.981581.641.721.641.650.053.31.581.721.101.32220104761.551.541.591.671.590.063.61.541.670.811.41220106702.302.272.272.222.260.031.42.222.301.351.06220305202.832.882.792.882.840.041.42.792.881.310.78220305264.254.554.554.854.550.245.44.254.851.221.22220307092.032.082.152.352.150.146.62.032.350.871.40220307102.092.132.132.202.140.052.12.092.201.041.36220313642.352.352.402.602.430.124.92.352.600.631.47220603472.262.322.372.532.370.124.92.262.530.951.38220305263.353.253.253.553.350.144.23.253.550.711.41220705821.791.771.761.811.780.021.31.761.810.911.28220711133.002.832.802.652.820.145.12.653.001.181.26220712691.962.052.122.242.090.125.61.962.241.131.25220712702.142.202.332.392.260.115.02.142.391.081.08220713082.042.011.972.042.010.031.61.972.041.340.83220713092.102.012.072.082.070.041.72.012.101.430.89220816931.961.891.942.001.950.052.41.892.001.281701.781.831.851.790.073.71.701.851.330.86格拉布斯检验结果表明测定结果中无异常值（当n=4，α=0.05时临界值为1.481，α=0.01时临界值为1.496）。各实验室试验数据表3实验室一试验数据样品编号氨基酸含量（%）液相法%系数f测定值1测定值2平均值相对偏差22070115.2913.6714.485.66.962.0822070226.6424.9325.783.312.812.0122070323.3922.2922.842.411.691.9522070417.8516.9217.392.79.381.8522070516.3217.8917.114.610.881.5722070613.0014.3513.674.98.971.5322070714.1113.2813.703.07.771.7622070814.0512.6713.365.29.051.4822070925.8324.0824.963.515.371.6222071024.6023.4524.032.413.541.7722071111.2210.3610.794.07.061.5322071218.6020.6119.605.110.161.9322071320.6422.0021.323.211.551.8522071421.1820.4920.841.711.521.8122071522.2521.0021.632.910.422.0822071625.9723.7424.854.512.262.032207179.249.299.270.36.631.4022071810.6711.4211.043.47.671.442207190.450.410.434.20.192.292207200.550.600.583.90.272.1222072110.809.9610.384.07.351.4122072213.2414.1013.673.19.901.3822072317.0415.1316.095.98.291.9422072416.8518.4317.644.59.721.812207258.8910.009.455.90.7412.752207263.002.732.864.81.092.6322072718.7120.5119.614.69.472.0722072823.5521.3822.464.810.472.1522072925.3224.3124.822.011.282.2022073015.6414.1714.914.97.681.9422073117.0918.6317.864.38.552.0922073222.6921.5422.112.69.842.2522073324.6726.1725.422.911.032.3122073422.7024.6123.664.014.781.6022073518.4517.1817.823.612.731.40220400259.3810.099.733.72.593.762204019230.5732.6031.593.27.694.112204023628.4330.3329.383.219.091.54220403602.292.142.213.30.653.43220407289.429.179.301.35.181.802205005725.0323.2924.163.615.401.572205020838.1734.5836.374.97.714.722205094515.1213.6414.385.28.761.642205143726.6729.9228.295.78.333.402205148121.2120.5320.871.612.331.692206034716.6215.5016.063.54.613.482205002316.3915.0815.744.211.391.382205002415.9017.3816.644.49.841.692205002517.4217.0317.231.111.411.512205002618.3216.4717.395.310.921.592205019331.9129.0430.484.715.222.002205019427.8627.0627.461.514.961.842205019530.3527.6529.004.614.871.952201047516.6715.2815.984.310.081.582201047616.2614.8015.534.710.001.552201067019.6020.8120.203.08.792.30220305202.943.303.125.91.102.83220305260.350.330.343.50.084.25220307090.370.340.353.70.172.032203071020.7219.4420.083.29.602.09220313642.422.242.333.80.992.352206034710.6310.2310.431.94.612.26220305260.250.280.274.50.083.352207058223.9726.5925.285.214.131.79220711130.580.620.603.30.203.00220712696.426.006.213.43.171.96220712706.386.556.471.33.022.142207130819.0818.3218.702.09.162.042207130917.5018.5018.002.88.582.102208169323.3325.5624.444.512.481.962208169426.6424.2525.444.714.961.70表4实验室二试验数据样品编号氨基酸含量（%）液相法%系数f测定值1测定值2平均值相对偏差22070113.8314.9014.373.76.962.0622070224.2626.4725.374.312.811.9822070321.7823.0522.422.811.691.9222070416.5717.6217.093.19.381.8222070518.1616.8417.503.810.881.6122070614.8813.2714.085.78.971.5722070714.6213.5214.073.97.771.8122070812.2413.4512.844.79.051.4222070924.9623.6124.282.815.371.5822071024.0725.0424.562.013.541.8122071110.1611.2210.694.97.061.5122071218.0019.4218.713.810.161.8422071321.0022.2721.642.911.551.8722071420.8421.8221.332.311.521.8522071522.6720.5821.634.810.422.0822071623.5125.0324.273.112.261.982207179.5710.039.802.36.631.4822071810.0811.1710.635.17.671.392207190.430.390.414.50.192.202207200.530.560.553.30.272.0022072110.2411.0810.663.97.351.4522072214.4813.7114.102.79.901.4222072315.3115.9515.632.08.291.8822072419.6317.8718.754.79.721.932207259.548.529.035.60.7412.192207262.912.642.774.91.092.5422072717.9919.3718.683.79.471.9722072822.2823.9123.103.510.472.2122072924.8623.2124.043.411.282.1322073014.8116.3715.595.07.682.0322073117.0116.4116.711.88.551.9622073221.6920.5921.142.69.842.1522073324.6725.8925.282.411.032.2922073424.1022.1723.134.214.781.5722073517.4519.0918.274.512.731.44220400259.918.939.425.22.593.642204019229.7032.1630.934.07.694.022204023630.5027.5229.015.119.091.52220403602.182.422.305.30.653.56220407289.829.309.562.85.181.852205005723.5724.0523.811.015.401.552205020835.1939.0837.145.27.714.822205094514.3013.0613.684.58.761.562205143728.1730.0829.133.38.333.502205148119.5020.5820.042.712.331.632206034717.3415.5516.455.54.613.572205002317.6215.5716.606.211.391.462205002417.4616.4816.972.99.841.732205002517.9716.8817.423.111.411.532205002615.8817.3116.604.310.921.522205019331.5728.6130.094.915.221.982205019429.9127.1328.524.914.961.912205019526.8730.1728.525.814.871.922201047517.3215.7716.544.710.081.642201047614.3916.4215.416.610.001.542201067020.5619.2719.923.28.792.27220305203.033.303.174.31.102.88220305260.380.340.365.50.084.55220307090.370.360.361.20.172.082203071021.2819.6020.444.19.602382.282.332.10.992.352206034711.1710.2310.704.44.612.32220305260.250.270.264.60.083.252207058226.2723.6524.965.214.131.77220711130.560.570.570.90.202.83220712696.836.176.505.13.172.05220712706.476.816.642.63.022.202207130817.8218.9918.403.29.162.012207130916.9217.6717.292.28.582.012208169322.5624.5323.554.212.481.892208169425.4927.6126.554.014.961.78表5实验室三试验数据样品编号氨基酸含量（%）液相法%系数f测定值1测定值2平均值相对偏差22070113.1014.1613.633.96.961.9622070227.3224.9426.134.512.812.0422070323.9023.1423.521.611.692.0122070417.1815.9516.573.79.381.7722070515.6117.1916.404.810.881.5122070613.5412.2812.914.98.971.4422070712.7013.5213.113.17.771.6922070813.3612.8413.102.09.051.4522070924.7226.6225.673.715.371.6722071024.8723.5424.202.713.541.792207119.6310.7610.205.57.061.4422071218.6219.7819.203.010.161.8922071321.5523.1822.363.711.551.9422071421.4119.4520.434.811.521.7722071522.1720.4221.294.110.422.0422071624.2722.2423.264.412.261.902207179.399.759.571.96.631.4422071812.0011.0011.504.37.671.502207190.390.420.413.40.192.182207200.520.570.555.00.272.0022072111.6310.5211.085.07.351.5122072215.1414.3814.762.69.901.4922072315.6814.4915.083.98.291.8222072418.8019.6319.212.29.721.982207258.989.729.354.00.7412.632207263.182.913.054.51.092.7922072718.6817.2217.954.19.471.9022072820.5722.6521.614.810.472.0622072922.0223.0322.522.211.282.0022073014.8113.8114.313.57.681.8622073118.3816.7517.564.68.552.0622073220.9619.5020.233.69.842.0622073325.8924.6725.282.411.032.2922073423.2221.7322.473.314.781.5222073519.0017.5518.274.012.731.442204002510.009.209.604.22.593.712204019230.7529.9330.341.47.693.952204023632.0730.3331.202.819.091.63220403602.182.342.263.60.653.50220407289.478.689.084.35.181.752205005723.7321.9022.824.015.401.482205020837.6335.0436.343.67.714.712205094512.6414.0513.355.38.761.522205143730.5028.7529.633.08.333.562205148120.0818.7519.423.412.331.572206034716.0915.7815.941.04.613.462205002316.3915.9016.151.511.391.422205002419.1818.1118.652.99.841.902205002516.8015.2316.024.911.411.402205002616.8115.0415.925.510.921.462205019330.2627.5728.914.715.221.902205019427.9129.7428.833.214.961.932205019528.9626.3527.654.714.871.862201047516.6717.9717.323.810.081.722201047616.3415.3715.853.110.001.592201067019.6020.3219.961.88.792.27220305203.123.033.071.51.102.79220305260.370.350.363.30.084.55220307090.360.390.374.70.172.152203071021.1219.8420.483.19.602482.282.384.20.992.402206034711.4110.4710.944.34.612.37220305260.260.250.261.50.083.252207058223.7326.0324.884.614.131.76220711130.530.590.565.40.202.80220712697.006.426.714.33.172.12220712706.817.247.033.13.022.332207130817.4818.6618.073.39.161.972207130917.5018.0817.791.68.582.072208169324.9623.5024.233.012.481.942208169426.4628.4127.433.514.961.83表6实验室四试验数据样品编号氨基酸含量（%）液相法%系数f测定值1测定值2平均值相对偏差22070113.5912.2012.895.46.961.8522070224.4326.8125.624.612.812.0022070322.3723.5622.972.611.691.9622070418.5817.4518.023.29.381.9222070516.6718.2517.464.510.881.6022070612.8213.9913.404.38.971.5022070713.1611.8812.525.17.771.6122070814.3113.1013.714.49.051.5122070925.4323.5324.483.915.371.5922071025.8424.2525.043.213.541.852207119.969.049.504.97.061.3522071218.9817.1918.094.910.161.7822071321.3622.9122.143.511.551.9222071418.6319.8619.243.211.521.6722071521.6723.2522.463.510.422.1622071624.5223.4223.972.312.261.9622071710.4910.1210.311.86.631.5622071812.0810.9211.505.17.671.502207190.440.480.464.10.192.452207200.530.500.512.70.271.8822072112.0111.3611.682.87.351.5922072213.7112.8613.293.29.901.3422072314.9513.9414.453.58.291.7422072418.8017.2218.014.49.721.852207259.268.528.894.20.7412.002207262.823.092.954.61.092.7122072717.4719.2818.384.99.471.9422072822.0120.1221.074.510.472.0122072924.1323.3023.721.711.282.1022073014.1712.9913.584.47.681.7722073118.8919.6619.272.08.552.2622073220.1418.5919.364.09.841.972207