美国药典USP32-重金属测试
2020版《中国药典》重金属检验操作规程(USP)
一、目的：制订详尽的工作程序，规范检验操作，保证检验数据的准确性。
二、范围：本标准适用于参考美国药典标准检验品种重金属的测定。
三、职责：1、检验员：严格按操作规程操作，认真、及时、准确地填写检验记录；2、化验室负责人：监督检查检验员执行本操作规程。
四、内容：1、特殊试剂:1.1硝酸铅原液:将159.8毫克的硝酸铅溶于100毫升水中，加入1毫升硝酸，然后用水稀释至1000毫升。
制备此溶液并将其储存在无可溶性铅盐的玻璃容器中。
1.2标准铅溶液:临用新制，用水稀释10.0毫升硝酸铅原液至100.0毫升。
每毫升标准铅溶液含有相当于10微克的铅。
以每克被测物质100微升标准铅溶液为基础制备的对比溶液包含相当于每百万份被测物质1部分的铅。
2、方法一:2.1 pH3.5乙酸盐缓冲液:溶解25克醋酸铵在25毫升水中，加入6mol/l盐酸38毫升。
如果需要调节，可用6mol/l氢氧化铵或6mol/l盐酸调节pH值为3.5，用水稀释至100毫升，并混合。
2.2标准制备:将标准铅溶液（20微克铅）2毫升放入50毫升比色管中，用水稀释至25毫升。
使用pH计或短程pH指示纸作为外部指示剂，用1mol/l乙酸或6mol/l氢氧化铵调节到3.0到4.0之间的pH，用水稀释至40毫升，混匀。
2.3供试品制备:按照各专著的指示，将试验准备的溶液放入50mL比色管中，或使用各专著中指定体积的酸，溶于水中，用水稀释至25mL，单位为按公式计算的待测物质：2.0/（1000L）其中L是重金属限度，占百分数。
使用pH计或短程pH指示剂纸作为外部指示剂，用1mol/l 乙酸或6 mol/l氢氧化铵调节pH值在3-4之间，用水稀释至40毫升，并混合。
2.4 监测制备:在第三根50mL比色管中，放入按供试品制备指示制备的溶液25mL，并加入2.0mL标准铅溶液。
使用pH计或短程pH指示剂纸作为外部指示剂，用1mol/l乙酸或6mol/l氢氧化铵调节pH值在3-4之间，用水稀释至40毫升，并混合。
(完整版)USP231重金属
USP<231>重金属本检验用以证实供试品中与硫离子作用显色的金属杂质含量，在规定检验条件下，不超过个论中规定的供试品重金属限度，以铅百分含量（重量比）计，与用标准铅溶液配制的对照进行目测比较（参看分光光度法和光散射法<851>中操作步骤部分的视觉比较）测定。
[注意：与本检验起反应的代表性物质为铅、汞、铋、砷、锑、锡、镉、银、铜和钼。
]除个论另有规定外，用方法I测定重金属含量。
方法I用于规定条件下可生成无色的、透明的制品的物质。
方法II适用于在方法I规定条件下无法生成无色透明制品的物质，或由于自身复杂特性，会对硫离子生成的金属沉淀造成干扰的物质，以及不挥发油和挥发油。
方法III湿消化法，仅在方法I和方法II均不适用时使用。
专用试剂硝酸铅贮备液：取硝酸铅159.8 mg，加已加硝酸1 mL的水100 mL，溶解，加水稀释至1000 mL。
于不含可溶铅盐的玻璃容器中配制和贮存该溶液。
标准铅溶液：使用当天配制。
取硝酸铅贮备液10.0 mL，加水稀释至100.0 mL。
每mL标准铅溶液相当于10 µg的铅。
以每g供试品100µL标准铅溶液为基准制备的参比溶液相当于每一百万份供试品中含有一份铅。
方法IpH 3.5醋酸盐缓冲液：取醋酸铵25.0 g，加水25 mL，溶解，加6 mol/L盐酸38.0 mL。
如有需要，用6 mol/L氨水或6 mol/L盐酸调节pH 为3.5，用水稀释至100 mL。
混匀。
标准溶液：用移液管取标准铅溶液2 mL（20 µg的铅），置50 mL比色管中，用水稀释至25 mL。
使用pH计或窄范围pH试纸作外部指示剂，用1mol/L醋酸或6mol/L氨水调节pH至3.0-4.0，用水稀释至40 mL，混匀。
供试溶液：取如个论所述制备的供试液25 mL，置50 mL比色管中；或当个论另有规定时，使用个论中规定体积量的酸溶解，并用水稀释至25 mL。
美国药典凡例中英文 USP32 NF27 General Notice
4. MONOGRAPHS AND GENERAL CHAPTERS 正文和附录
4.10 Monographs 专论 Monographs set forth the article's name, definition, specification, and other requirements related to packaging, storage, and labeling. The specification consists of tests, procedures, and acceptance criteria that help ensure the identity, strength, quality, and purity of the article. For general requirements relating to specific monograph sections, see section 5, Monograph Components. 专论阐述了药品的名称，定义，质量标准，及其他和包装，储存和标签相关的要求。质量标 准包括测试，过程，及可接受范围，保证了药品的特性，规格，质量和纯度。对于特定专论 相关的一般要求，参见章节 5“专论构成”。 Because monographs may not provide standards for all relevant characteristics, some official substances may conform to the USP or NF standard but differ with regard to nonstandardized properties that are relevant to their use in specific preparations. To assure interchangeability in such instances, users may wish to ascertain functional equivalence or determine such characteristics before use. 由于专论可能无法提供全部特性的标准，有些药物可能符合 USP 或 NF 标准，但是其在特 定的制备中使用的相关非标准性质可能有所不同。为确保这个情况的互通性，使用者在使用 前须确定其功能对等或确定其特征。 4.10.10 Applicability of Test Procedures 测试过程的适用性 A single monograph may include several different tests, procedures, and/or acceptance criteria that reflect attributes of different manufacturers' articles. Such alternatives may be presented for different polymorphic forms, impurities, hydrates, and dissolution cases. Monographs indicate the
美国药典重金属检测方法-中文
铑
10
40
0.0001
钌
10
40
0.0002
铬
25
100
0.002
钼
25
100
0.0002
镍
25
100
0.002
钒
25
100
0.005
铜
250
1000
0.002
镁
250
1000
0.001
样品制备
范围广泛的各种样品都可以用 USP<232>/<233> 进行分 析，所以提供适合所有样品类型的详细样品处理方法并不现 实。有些药物样品可以直接分析（不用溶解），而其他样品 可以用水性溶剂（如水或稀酸）或适当的有机溶剂（如 2-丁 氧乙醇 : 水（25 : 75）[3]，DMSO 或 DGME）简单稀释或 溶解进行制备。用水性或有机溶剂进行简单稀释或溶解的 方法必须考虑样品的化学稳定性，并且对于有机溶剂溶解， 还要考虑样品中组分化合物的不同挥发性。对许多 API 来 说，用有机溶剂稀释是首选方法，这种情况下有必要采取有 助于稳定分析物的方法，以避免因较高或较低挥发性（与校 正标准品相比）成分的存在而造成的回收率波动 [7]。
USP<232> 包括一个涉及元素形态的章节，指出 As 和 Hg 的某些形态值得关注，因为其毒性比其它形态要大得 多。As 的 PDE 是指无机 As，如果总 As 浓度超出限度， 必须用一种能够对不同 As 形态进行分离和定量的方法对样 品进行重新分析。这样做的原因是无机 As 比常见的有机形 式（如，砷甜菜碱）毒性大得多，因此形态分析必须能够分 离其不同化学形态，确定无机 As（亚砷酸盐（三价 As）和 砷酸盐（四价 As））的总量低于限量。同样，Hg 限量也是 指无机 Hg（Hg2+），虽然甲基汞（MeHg）是毒性更大的 形态，但通常认为药物中不可能存在 MeHg。但如果样品来 自于可能含有相当量甲基汞的原料（如，鱼组织），也必须 对其进行特别的分离和测定。
USP232-233重金属检测方法-中文
铑
10
40
0.0001
钌
10
40
0.0002
铬
25
100
0.002
钼
25
100
0.0002
镍
25
100
0.002
钒
25
100
0.005
铜
250
1000
0.002
镁
250
1000
0.001
样品制备
范围广泛的各种样品都可以用 USP<232>/<233> 进行分 析，所以提供适合所有样品类型的详细样品处理方法并不现 实。有些药物样品可以直接分析（不用溶解），而其他样品 可以用水性溶剂（如水或稀酸）或适当的有机溶剂（如 2-丁 氧乙醇 : 水（25 : 75）[3]，DMSO 或 DGME）简单稀释或 溶解进行制备。用水性或有机溶剂进行简单稀释或溶解的 方法必须考虑样品的化学稳定性，并且对于有机溶剂溶解， 还要考虑样品中组分化合物的不同挥发性。对许多 API 来 说，用有机溶剂稀释是首选方法，这种情况下有必要采取有 助于稳定分析物的方法，以避免因较高或较低挥发性（与校 正标准品相比）成分的存在而造成的回收率波动 [7]。
元素 镉 铅 无机砷 无机汞 铱 锇 钯 铂 铑 钌 铬 钼 镍 钒 铜 镁
每日剂量 PDE（µg/日） 5 10 15 15 100 100 100 100 100 100 250 250 250 250 2500 2500
虽然 USP<232> 中制定的 PDE 限度用任何 USP<233> （ICP-OES 或 ICP-MS）中参考的仪器技术都可以测定 [6]， 但许多新药中使用越来越复杂而珍贵的原料药，可能只包 含非常少的量。对这些毫克级样品进行大比例稀释制备， 意味着使用检测限尽可能低的仪器非常重要。低检测限和 宽动态范围线性校正（安捷伦 7700 系列达到 9 个数量级） 是 ICP-MS 最非常可贵的特性。低检测限对于 USP<232> 要求必须以最低限量进行控制的潜在毒性痕量元素（特别是 As、Cd、Hg 和 Pb）尤为重要。
(完整版)USP231重金属
USP<231>重金属本检验用以证实供试品中与硫离子作用显色的金属杂质含量，在规定检验条件下，不超过个论中规定的供试品重金属限度，以铅百分含量（重量比）计，与用标准铅溶液配制的对照进行目测比较（参看分光光度法和光散射法<851>中操作步骤部分的视觉比较）测定。
[注意：与本检验起反应的代表性物质为铅、汞、铋、砷、锑、锡、镉、银、铜和钼。
]除个论另有规定外，用方法I测定重金属含量。
方法I用于规定条件下可生成无色的、透明的制品的物质。
方法II适用于在方法I规定条件下无法生成无色透明制品的物质，或由于自身复杂特性，会对硫离子生成的金属沉淀造成干扰的物质，以及不挥发油和挥发油。
方法III湿消化法，仅在方法I和方法II均不适用时使用。
专用试剂硝酸铅贮备液：取硝酸铅159.8 mg，加已加硝酸1 mL的水100 mL，溶解，加水稀释至1000 mL。
于不含可溶铅盐的玻璃容器中配制和贮存该溶液。
标准铅溶液：使用当天配制。
取硝酸铅贮备液10.0 mL，加水稀释至100.0 mL。
每mL标准铅溶液相当于10 µg的铅。
以每g供试品100µL标准铅溶液为基准制备的参比溶液相当于每一百万份供试品中含有一份铅。
方法IpH 3.5醋酸盐缓冲液：取醋酸铵25.0 g，加水25 mL，溶解，加6 mol/L盐酸38.0 mL。
如有需要，用6 mol/L氨水或6 mol/L盐酸调节pH 为3.5，用水稀释至100 mL。
混匀。
标准溶液：用移液管取标准铅溶液2 mL（20 µg的铅），置50 mL比色管中，用水稀释至25 mL。
使用pH计或窄范围pH试纸作外部指示剂，用1mol/L醋酸或6mol/L氨水调节pH至3.0-4.0，用水稀释至40 mL，混匀。
供试溶液：取如个论所述制备的供试液25 mL，置50 mL比色管中；或当个论另有规定时，使用个论中规定体积量的酸溶解，并用水稀释至25 mL。
甜菜碱盐酸盐USP32检测方法
甜菜碱盐酸盐USP32检测方法以及标准根据客户提供的资料，翻译情况如下：无水基础上测得甜菜碱盐酸盐含量：98%-100.5%包装和储存：密闭性好的容器美国药典USP标准参考：鉴别：A，红外线吸收（197）B，1比20的溶液符合氯化物的检测要求PH(791)： PH 值应为0.8-1.2之间，溶液比为1：10水份测定，方法1（921）：不超过0.5%炽灼残渣（281）：不超过0.1%重金属（231）：0.001%主含量：转移400mg精确称重过的甜菜碱盐酸盐到一个锥形瓶，加50ml冰乙酸，加热并搅拌直到溶液完全溶解，加入50ml乙酸汞检测溶液，冷的，加2滴结晶紫检测溶液，然后用0.1当量浓度的高氯酸滴定液滴定至一个绿色结点，进行一个空白对照，并做一些必要修正，每ml0.1当量浓度高氯酸相当于15.36mg甜菜碱盐酸盐。
<197> 分光光度鉴别检验分光光度法检测对于很多药典化学物质的鉴别做出了意味深远的贡献。
下面的这些步骤适用于那些吸收红外和/或者紫外线的物质（见分光光度测定法和光散射<851>）。
一个物质的红外吸收光谱，在与从对应的USP标准品处获得的光谱图进行比较后，或许提供了从任何单一检验中所能获得的关于该物质的鉴别的最具决定性的证据。
而另一方面，紫外吸收图谱则并未展示出高度的特异性。
与红外吸收和紫外吸收检验标准的符合性，如在很大比例的药典专论中所要求的，用于供试样品的鉴别几乎不会导致任何质疑。
红外吸收光谱六种方法用于已干燥的待检样品和对照品的前处理。
在专论中提到<197K>意味着该待检物质与溴化钾充分混合。
在专论中提到<197M>意味着该待检物质被精细地碾磨并分散于矿物油中。
在专论中提到<197F>表示待检样品均匀地悬置于适当的（例如，氯化钠或溴化钾）压片板之间。
在专论中提到<197S>表示使用具体专论中指定的溶剂中并按照指定的浓度配制了溶液，并且如果具体的专论中没有指定不同的单位通道长度，则该溶液在0.1-mm的单位中检测。
硫酸软骨素质量标准 及含量测定
硫酸软骨素质量标准及含量测定青岛大学药学系高华gaohuaqy@一、硫酸软骨素结构是种 硫酸软骨素(Chondroitin Sulfate，CS)，是一种重要的天然酸性粘多糖。
硫酸软骨素是由D-葡糖醛酸和N-乙酰基-D-氨基半乳糖以β-1,3糖苷键结合而成的双糖，而双糖之间以β-1,4苷键连接而成生物大分子。
-14CS相对分子质量一般为5000～50000Da。
根据硫酸基在半乳糖上位置不同主要分为硫酸软骨素A(CSA)硫酸软骨素C(CSC)硫酸软骨素结构根据硫酸基在半乳糖上位置不同主要分为硫酸软骨素A(CSA)和硫酸软骨素C(CSC)硫酸软骨素A（CSA）硫酸软骨素C（CSC）硫酸基团在半乳糖的第4位C上硫酸基团在半乳糖的第6位C上二硫酸软骨素存在形式二、硫酸软骨素存在形式ChS的来源有陆产动物和海洋动物两大类。
陆产动物主要是牛、猪、羊和禽等软骨组织中，如喉骨、鼻中骨、气管、血管壁等，主要成分4-硫酸软骨素(CSA) 。
海洋动物包括软骨鱼的软骨、海参和贝类等，主要成分6-硫酸软骨素(CSC)。
贝类等主要成分6软骨中的硫酸软骨素与蛋白质相结合以蛋白多糖的形式存在组织中。
三、硫酸软骨素质量标准z USP32--美国药典29版硫酸软骨素（牛、猪、鸡、鲨鱼）z JPC1997 --《日本药局方外标准》(1997)硫酸软骨素（牛、猪、鲨鱼）硫酸软骨素（牛猪鲨鱼）z WS-10001-(HD-0894)-2002硫酸软骨素A 钠z WS-10001-(HD-0892)-2002 硫酸软骨素（供注射用）z WS-10001-(HD-0895)-2002 硫酸软骨素A钠胶囊WS-10001-(HD-0896)-2002 硫酸软骨素滴眼液WS-10001-(HD-0854)-2002 复方硫酸软骨素片硫酸软骨素(USP32/JPC1997)z ：白色至乳白色z：牛、猪、鸡软骨◎硫酸软骨素钠◎澄清度无色透明外观白色乳白色z 理化指标来源牛猪鸡软骨◎硫酸软骨素钠：≥90% ◎澄清度：无色透明◎pH 值(1%溶液)：5.5～7.5 ◎干燥失重：≤10.0% 氯化物硫酸盐◎氯化物：≤0.142% ◎硫酸盐：≤0.240%◎重金属：≤20 mg/kg ◎积灼残渣：23.0～30.0% ◎旋光度：-28°～-32°◎氮含量：2.5～3.8%z 微生物指标◎细菌总数：≤100 CFU/g ◎霉菌和酵母：≤10 CFU/g 微物指标◎沙门氏菌，大肠杆菌，葡萄球菌，致病菌：不得检出硫酸软骨素(USP32/JPC1997)z ：白色至乳白色z：鲨鱼软骨◎硫酸软骨素钠◎澄清度无色透明外观白色乳白色z 理化指标来源鲨软骨◎硫酸软骨素钠：≥90% ◎澄清度：无色透明◎pH 值(1%溶液)：5.5～7.5 ◎干燥失重：≤10.0% 氯化物硫酸盐◎氯化物：≤0.142% ◎硫酸盐：≤0.240%◎重金属：≤20 mg/kg ◎积灼残渣：23.0～30.0% ◎旋光度：-12°～-18°◎氮含量：2.5～3.8%z 微生物指标◎细菌总数:≤100 CFU/g ◎霉菌和酵母:≤10 CFU/g 微物指标◎沙门氏菌，大肠杆菌，葡萄球菌，致病菌：不得检出硫酸软骨素A 钠猪软骨z外观：白色或微黄色外白色或微黄色z来源：猪软骨z理化指标◎硫酸软骨素钠：≥90% ◎澄清度：无色透明◎pH值(1%溶液)：5.57.5 ◎干燥失重：≤5.0%～◎硫酸盐：与标准硫酸钾溶液5.0ml对照液比较，不得更浓(0.5%)◎重金属：≤20 mg/kg ◎平均分子量：35000500035000-5000 ◎旋光度：-27°～-33°◎氮含量：2.2～3.8%z微生物指标◎细菌总数:≤1000 CFU/g ◎霉菌和酵母:≤100 CFU/g ◎沙门氏菌，大肠杆菌，葡萄球菌，致病菌：不得检出硫酸软骨素（供注射用）猪软骨z外观：白色或微黄色外白色或微黄色z来源：猪软骨z理化指标◎硫酸软骨素钠：≥90% ◎澄清度(5%溶液): A≤0.05640nm◎pH值(1%溶液)：5.57.5 ◎干燥失重：≤10.0%～◎硫酸盐：与标准硫酸钾溶液5.0ml对照液比较，不得更浓(0.5%)≤20 mg/kg ◎氯化物：≤1.0%◎重金属：10%◎旋光度：-27°～-33°◎氮含量：2.5～3.8%z微生物指标◎细菌总数:≤1000 CFU/g ◎霉菌和酵母:≤100 CFU/g ◎沙门氏菌，大肠杆菌，葡萄球菌，致病菌：不得检出四、硫酸软骨素含量测定方法z各个国家对CS含量测定方法不尽相同。
美国药典USP32-重金属测试
<231> 重金属本试验系在规定的试验条件下，金属离子与硫化物离子反应显色，通过制备的标准铅溶液目视比较测定，以确证供试品中重金属杂质含量不超过各论项下规定的限度（以供试品中铅的百分比表示，以重量计）。
（见分光光度法和光散射项下测定法目视比较法<851>）[ 注意:对本试验有响应的典型物质有铅、汞、铋、砷、锑、锡、镉、银、铜和钼等]。
除各论另有规定外，按第一法测定重金属。
第一法适用于在规定试验条件下，能产生澄清、无色溶液的物质。
第二法适用于在第一法规定试验条件下不能产生澄清、无色溶液的物质，或者适用于由于性质复杂，易干扰硫化物离子与金属离子形成沉淀的物质，或者是不易挥发的和易挥发的油类物质。
第三法为湿消化法，仅用于第一法、第二法都不适合的情况。
特殊试剂特殊试剂特殊试剂特殊试剂硝酸铅贮备液—取硝酸铅159.8mg，溶于100ml水中，加1ml硝酸，用水稀释至1000ml。
制备和贮存本溶液的玻璃容器应不含可溶性铅。
标准铅溶液—使用当天，取硝酸铅贮备液10.0ml，用水稀释至100.0ml。
每1ml的标准铅溶液含相当于10µg的铅。
按每克供试品取100µl标准铅溶液制备的对照溶液，相当于供试品含百万分之一的铅。
方法方法方法方法IIII pH3.5醋酸盐缓冲液—取醋酸铵25.0g溶于25ml水中，加6N盐酸液38.0ml，必要时，用6N氢氧化铵液或6N盐酸液调节pH至3.5，用水稀释至100ml，混匀。
标准溶液准备—精密量取标准铅溶液2ml，（相当于20µg的Pb），置50ml比色管中，加水稀释至25ml，以精密pH试纸作为外指示剂，用1N醋酸液或6N 氢氧化铵液调节pH至3.0~4.0，用水稀释至40ml，混匀。
供试品溶液制备—取各论项下规定的供试品溶液25ml，置50ml比色管中，或用各论项下规定用量的酸溶解样品，再用水稀释至25ml，供试品以g计，按下式计算： 2.0/（1000L）式中L是重金属限度（%）。
硫酸软骨素质量标准 及含量测定
硫酸软骨素质量标准及含量测定青岛大学药学系高华gaohuaqy@一、硫酸软骨素结构是种 硫酸软骨素(Chondroitin Sulfate，CS)，是一种重要的天然酸性粘多糖。
硫酸软骨素是由D-葡糖醛酸和N-乙酰基-D-氨基半乳糖以β-1,3糖苷键结合而成的双糖，而双糖之间以β-1,4苷键连接而成生物大分子。
-14CS相对分子质量一般为5000～50000Da。
根据硫酸基在半乳糖上位置不同主要分为硫酸软骨素A(CSA)硫酸软骨素C(CSC)硫酸软骨素结构根据硫酸基在半乳糖上位置不同主要分为硫酸软骨素A(CSA)和硫酸软骨素C(CSC)硫酸软骨素A（CSA）硫酸软骨素C（CSC）硫酸基团在半乳糖的第4位C上硫酸基团在半乳糖的第6位C上二硫酸软骨素存在形式二、硫酸软骨素存在形式ChS的来源有陆产动物和海洋动物两大类。
陆产动物主要是牛、猪、羊和禽等软骨组织中，如喉骨、鼻中骨、气管、血管壁等，主要成分4-硫酸软骨素(CSA) 。
海洋动物包括软骨鱼的软骨、海参和贝类等，主要成分6-硫酸软骨素(CSC)。
贝类等主要成分6软骨中的硫酸软骨素与蛋白质相结合以蛋白多糖的形式存在组织中。
三、硫酸软骨素质量标准z USP32--美国药典29版硫酸软骨素（牛、猪、鸡、鲨鱼）z JPC1997 --《日本药局方外标准》(1997)硫酸软骨素（牛、猪、鲨鱼）硫酸软骨素（牛猪鲨鱼）z WS-10001-(HD-0894)-2002硫酸软骨素A 钠z WS-10001-(HD-0892)-2002 硫酸软骨素（供注射用）z WS-10001-(HD-0895)-2002 硫酸软骨素A钠胶囊WS-10001-(HD-0896)-2002 硫酸软骨素滴眼液WS-10001-(HD-0854)-2002 复方硫酸软骨素片硫酸软骨素(USP32/JPC1997)z ：白色至乳白色z：牛、猪、鸡软骨◎硫酸软骨素钠◎澄清度无色透明外观白色乳白色z 理化指标来源牛猪鸡软骨◎硫酸软骨素钠：≥90% ◎澄清度：无色透明◎pH 值(1%溶液)：5.5～7.5 ◎干燥失重：≤10.0% 氯化物硫酸盐◎氯化物：≤0.142% ◎硫酸盐：≤0.240%◎重金属：≤20 mg/kg ◎积灼残渣：23.0～30.0% ◎旋光度：-28°～-32°◎氮含量：2.5～3.8%z 微生物指标◎细菌总数：≤100 CFU/g ◎霉菌和酵母：≤10 CFU/g 微物指标◎沙门氏菌，大肠杆菌，葡萄球菌，致病菌：不得检出硫酸软骨素(USP32/JPC1997)z ：白色至乳白色z：鲨鱼软骨◎硫酸软骨素钠◎澄清度无色透明外观白色乳白色z 理化指标来源鲨软骨◎硫酸软骨素钠：≥90% ◎澄清度：无色透明◎pH 值(1%溶液)：5.5～7.5 ◎干燥失重：≤10.0% 氯化物硫酸盐◎氯化物：≤0.142% ◎硫酸盐：≤0.240%◎重金属：≤20 mg/kg ◎积灼残渣：23.0～30.0% ◎旋光度：-12°～-18°◎氮含量：2.5～3.8%z 微生物指标◎细菌总数:≤100 CFU/g ◎霉菌和酵母:≤10 CFU/g 微物指标◎沙门氏菌，大肠杆菌，葡萄球菌，致病菌：不得检出硫酸软骨素A 钠猪软骨z外观：白色或微黄色外白色或微黄色z来源：猪软骨z理化指标◎硫酸软骨素钠：≥90% ◎澄清度：无色透明◎pH值(1%溶液)：5.57.5 ◎干燥失重：≤5.0%～◎硫酸盐：与标准硫酸钾溶液5.0ml对照液比较，不得更浓(0.5%)◎重金属：≤20 mg/kg ◎平均分子量：35000500035000-5000 ◎旋光度：-27°～-33°◎氮含量：2.2～3.8%z微生物指标◎细菌总数:≤1000 CFU/g ◎霉菌和酵母:≤100 CFU/g ◎沙门氏菌，大肠杆菌，葡萄球菌，致病菌：不得检出硫酸软骨素（供注射用）猪软骨z外观：白色或微黄色外白色或微黄色z来源：猪软骨z理化指标◎硫酸软骨素钠：≥90% ◎澄清度(5%溶液): A≤0.05640nm◎pH值(1%溶液)：5.57.5 ◎干燥失重：≤10.0%～◎硫酸盐：与标准硫酸钾溶液5.0ml对照液比较，不得更浓(0.5%)≤20 mg/kg ◎氯化物：≤1.0%◎重金属：10%◎旋光度：-27°～-33°◎氮含量：2.5～3.8%z微生物指标◎细菌总数:≤1000 CFU/g ◎霉菌和酵母:≤100 CFU/g ◎沙门氏菌，大肠杆菌，葡萄球菌，致病菌：不得检出四、硫酸软骨素含量测定方法z各个国家对CS含量测定方法不尽相同。
USP32
LeucineC6H13NO2 131.17l-Leucine.l-Leucine [61-90-5].» Leucine contains not less than 98.5 percent and not more than 101.5 percent of C6H13NO2, as l-leucine, calculated on the dried basis.Packaging and storage— Preserve in well-closed containers.USP Reference standards 11—USP L-Leucine RS.USP L-Valine RS.Identification, Infrared Absorption 197K.Specific rotation 781S: between +14.9 and +17.3.Test solution: 40 mg per mL, in 6 N hydrochloric acid.pH 791: between 5.5 and 7.0, in a solution (1 in 100).Loss on drying 731— Dry it at 105 for 3 hours: it loses not more than 0.2% of its weight.Residue on ignition 281: not more than 0.4%.Chloride 221— A 0.73-g portion shows no more chloride than corresponds to 0.50 mL of 0.020 N hydrochloric acid (0.05%).Sulfate 221— A 0.33-g portion shows no more sulfate than corresponds to 0.10 mL of 0.020 N sulfuric acid (0.03%).Iron 241: 0.003%.Heavy metals, Method II 231: 0.0015%.Chromatographic purity—Adsorbent: 0.25-mm layer of chromatographic silica gel mixture.Test solution— Dissolve an accurately weighed quantity of Leucine in 0.1 N hydrochloric acid to obtain a solution having a concentration of 10 mg per mL. Apply 5 µL.Standard solution— Dissolve an accurately weighed quantity of USPL-Leucine RS in 0.1 N hydrochloric acid to obtain a solution having a known concentration of about 0.05 mg per mL. Apply 5 µL. [note—This solution has a concentration equivalent to about 0.5% of that of the Test solution.]System suitability solution— Prepare a solution in 0.1 N hydrochloric acid containing 0.4 mg each of USP L-Leucine RS and USP L-Valine RS per mL. Apply 5 µL.Spray reagent— Dissolve 0.2 g of ninhydrin in 100 mL of a mixture of butyl alcohol and 2 N acetic acid (95:5).Developing solvent system— Prepare a mixture of butyl alcohol, glacial acetic acid, and water (60:20:20).Procedure— Proceed as directed for Thin-Layer Chromatography under Chromatography 621 . After air-drying the plate, spray with Spray reagent, and heat between 100 and 105 for about 15 minutes. Examine the plate under white light. The chromatogram obtained from the System suitability solution exhibits two clearly separated spots. Any secondary spot in the chromatogram obtained from the Test solution is not larger or more intense than the principal spot in the chromatogram obtained from the Standard solution: not more than 0.5% of any individual impurity is found; and not more than 2.0% of total impurities is found.Assay— Transfer about 130 mg of Leucine, accurately weighed, to a 125-mL flask, dissolve in a mixture of 3 mL of formic acid and 50 mL of glacial acetic acid, and titrate with 0.1 N perchloric acid VS, determining the endpoint potentiometrically. Perform a blank determination, and make any necessary correction. Each mL of 0.1 N perchloric acid is equivalent to 13.12 mg ofC6H13NO2.Auxiliary Information— Please check for your question in the FAQs before contacting USP.Topic/Question Contact Expert CommitteeMonograph Curtis Phinney1-301-816-8540 (DSN05) Dietary Supplements - Non-BotanicalsReference Standards Lili Wang, Technical Services Scientist1-301-816-8129RSTech@USP32–NF27 Page 2758Chromatographic Column—LEUCINEChromatographic columns text is not derived from, and not part of, USP 32 or NF 27.亮氨酸c6h13no2 131.17亮氨酸。
【英文版】USP32富马酸亚铁含量的测定
美国药典32版（USP32）英文版富马酸亚铁含量的测定C4H2FeO4169.902-Butenedioic acid, (E)-, iron(2+) salt. 【2-反丁烯二酸，（E）-亚铁盐】Iron(2+) fumarate 【富马酸亚铁】»Ferrous Fumarate contains not less than 97.0 percent and not more than 101.0 percent of C4H2FeO4, calculated on the dried basis.Packaging and storage— Preserve in well-closed containers.Identification【鉴别】—A: To 1.5 g add 25 mL of dilute hydrochloric acid (1 in 2). Dilute with water to 50 mL, heat to dissolve, then cool, filter on a fine-porosity【细孔径】, sintered-glass crucible【烧结玻璃坩埚】, wash the precipitate【沉淀】with dilute hydrochloric acid (3 in 100), saving the filtrate for Identification test B, and dry the precipitate at 105℃: the IR absorption【外红吸收】of a potassium bromide【溴化钾】dispersion of the dried precipitate so obtained exhibits maxima 【最大值】only at the same wavelengths as that of a similar preparation of USP Fumaric Acid RS.B: A portion of the filtrate obtained in the preceding test responds to the tests for Iron<191>.Loss on drying <731>— Dry it at 105℃for 16 hours: it loses not more than 1.5% of its weight. Sulfate【硫酸盐】—Transfer【移取】1.0 g to a 250-mL beaker【烧杯】, add 100 mL of water, and heat on a steam bath【蒸气浴】, adding hydrochloric acid dropwise【逐滴加入盐酸】, until complete solution 【溶液】is effected (about 2 mL of the acid will be required).Filter【过滤】the solution if necessary, and dilute【稀释】the filtrate【滤液】with water to 100 mL. Heat the filtrate to boiling【沸腾】,add 10 mL of barium chloride TS【氯化钡试液，TS=Test Solution 试液】, warm on a steam bath for 2 hours, cover, and allow to stand for【静置】16 hours. (If crystals【结晶】of ferrous fumarate form【形成】, warm the solution on the steam bath to dissolve【溶解】them.)Pass the solution through ashless filter paper【无灰滤纸】, wash the residue【滤渣】with hot water until, with the addition of ammonium sulfide TS【硫化铵试液，TS=Test Solution 试液】, a black precipitate【沉淀】is no longer formed in the filtrate, and transfer the paper containing the residue to a tared crucible【已称重的坩埚】.Char【碳化】the paper, without burning, and ignite the crucible【灼烧坩埚】and its contents at 600℃to constant weight【恒重】: each mg of residue【残留物】is equivalent to【相当于】0.412 mg of SO4. Not more than 0.2% is found.Arsenic, Method I <211>— Transfer 2.0 g to a beaker, and add 10 mL of water and 10 mL of sulfuric acid. Warm to precipitate the fumaric acid completely, cool, add 30 mL of water, and filter into a 100-mL volumetric flask【容量瓶】. Wash the precipitate with water, adding the washings to the flask, add water to volume, and mix. Transfer 50.0 mL of this solution into the arsine generator flask【砷化氢发生器】, and dilute with water to 55 mL: the resulting solution meets the requirements of the test, the addition of 20 mL of 7 N sulfuric acid specified【指定】for Procedure being omitted【省略】. The limit is 3 ppm.Limit of ferric iron— Transfer 2.0 g, accurately weighed【精确称量】, to a glass-stoppered【玻璃塞】, 250-mL conical flask【锥形瓶】, add 25 mL of water and 4 mL of hydrochloric acid, and heat on a hot plate【电炉】until solution is complete. Insert the stopper in the flask, and cool to room temperature. Add 3 g of potassium iodide【碘化钾】, insert the stopper in the flask, swirl to mix【摇匀】, and allow to stand in the dark for 5 minutes. Remove the stopper, add 75 mL of water, and titrate with 0.1 N sodium thiosulfate VS【硫代硫酸钠滴定液】, adding 3 mL of starch TS【淀粉溶液】as the end-point is approached【临近终点】. Not more than 7.16 mL of 0.1 N sodium thiosulfate is consumed (2.0%).Limit of lead— [note—For the preparation of all aqueous solutions【水溶液】and for the rinsing【冲洗】of glassware before use, employ water【用水】that has been passed through a strong-acid【强酸】, strong-base【强碱】, mixed-bed ion-exchange resin【混合床离子交换树脂】before use. Select all reagents【试剂】to have as low a content of lead as practicable, and store all reagent solutions in containers of borosilicate glass【硼硅酸盐玻璃】. Clean glassware before use by soaking【浸泡】in warm 8 N nitric acid for 30 minutes and by rinsing with deionized water【去离子水】.]Ascorbic acid【抗坏血酸】–sodium iodide solution【碘化钠溶液】— Dissolve 20 g of ascorbic acid and 38.5 g of sodium iodide in water in a 200-mL volumetric flask, dilute with water to volume, and mix.Trioctylphosphine oxide solution【三正辛基氧膦溶液】— [Caution—This solution causes irritation【刺激】. Avoid contact with eyes, skin, and clothing. Take special precautions in disposing of unused portions of solutions to which this reagent is added. ] Dissolve 5.0 g of trioctylphosphine oxide in 4-methyl-2-pentanone【4-甲基-2-戊酮】in a 100-mL volumetric flask, dilute with the same solvent to volume, and mix.Standard solution and Blank— Transfer 5.0 mL of Lead Nitrate Stock Solution【硝酸铅储备液】, prepared as directed in the test for Heavy Metals <231>, to a 100-mL volumetric flask, dilute with water to volume, and mix. Transfer 2.0 mL of the resulting solution to a50-mL beaker. To this beaker and to a second, empty beaker (Blank) add 6 mL of nitric acid and 10 mL of perchloric acid【高氯酸】, and evaporate【蒸发】in a hood【橱】to dryness. [Caution—Use perchloric acid in a well-ventilated【通风良好】fume hood【通风橱】with proper precautions【适当的预防措施】. ] Cool, dissolve the residues in 10 mL of 9 N hydrochloric acid, and transfer with the aid of about 10 mL of water to separate 50-mL volumetric flasks. To each flask add 20 mL of Ascorbic acid–sodium iodide solution and 5.0 mL of Trioctylphosphine oxide solution, shake for 30 seconds, and allow to separate. Add water to bring the organic solvent layer into the neck of each flask, shake again, and allow to separate. The organic solvent layers are the Blank and the Standard solution, and they contain 0.0 and 2.0 µg of lead per mL, respectively.Test solution— Add 1.0 g of Ferrous Fumarate to a 50-mL beaker, and add 6 mL of nitric acid and 10 mL of perchloric acid. [Caution—Use perchloric acid in a well-ventilated fume hood with proper precautions. ] Cover with a ribbed【棱纹】watch glass【表面皿】, and heat in a hood until completely dry. Cool, dissolve the residue in 10 mL of 9 N hydrochloric acid, and transfer with the aid of about 10 mL of water to a 50-mL volumetric flask. Add 20mL of Ascorbic acid–sodium iodide solution and 5.0 mL of Trioctylphosphine oxide solution, shake for 30 seconds, and allow to separate. Add water to bring the organic solvent layer into the neck of the flask, shake again, and allow to separate. The organic solvent layer is the Test solution.Procedure【步骤】— Concomitantly【伴随，同时】determine the absorbances of the Blank, Standard solution, and Test solution at the lead emission line【铅发射线】at 283.3 nm with a suitable atomic absorption spectrophotometer (see Spectrophotometry and Light-scattering <851>) equipped with a lead hollow-cathode lamp【铅空心阴极灯】and an air–acetylene flame【空气-乙炔火焰】, using the blank to set the instrument to zero. In a suitable analysis, the absorbance of the Standard solution and the absorbance of the Blank are significantly different: the absorbance of the Test solution does not exceed that of the Standard solution (0.001%).Mercury【汞】— [notes—(1) Carry out【完成】this procedure in subdued light【避光】, since mercuric dithizonate【双硫腙汞】is light-sensitive【对光敏感】. (2) For preparation of solutions, see Mercury <261>.] Dissolve about 1 g, accurately weighed, in 30 mL of dilute nitric acid (1 in 10), with the aid of heat, on a steam bath. Cool quickly by immersion in an ice bath, and pass through a fine-porosity filter that previously has been washed with dilute nitric acid (1 in 10) and water. To the filtrate add 20 mL of sodium citrate solution【柠檬酸钠溶液】(1 in 4) and 1 mL of Hydroxylamine Hydrochloride Solution【盐酸羟胺溶液】.Prepare a control solution【对照溶液】consisting of 3.0 mL of Standard Mercury Solution, 30 mL of dilute nitric acid (1 in 10), 5 mL of sodium citrate solution (1 in 4), and 1 mL of Hydroxylamine Hydrochloride Solution.Using ammonium hydroxide【氨水】, adjust the control solution to a pH of 1.8, determined potentiometrically【电位滴定，pH计】, and transfer to a separator【分离器】. Using sulfuric acid, adjust the test solution to a pH of 1.8, determined potentiometrically, and transfer to a separator. Treat the solution under test and the control solution in parallel 【平行】as follows. Extract with two 5-mL portions of Dithizone Extraction Solution【双硫腙提取液】and 5 mL of chloroform【三氯甲烷】, pooling【收集】the chloroform extracts in a second separator. Add 10 mL of dilute hydrochloric acid (1 in 2), shake, allow the layers to separate, and discard the chloroform layer. Wash the acid extract with 3 mL of chloroform, and discardthe washing. Add 0.1 mL of edetate disodium solution【乙二胺四乙酸二钠溶液】(1 in 50) and 2 mL of 6 N acetic acid【乙酸】, mix, and add slowly 5 mL of ammonium hydroxide. Close the separator, cool it under cold running water, and dry its outer surface. Remove the stopper, and pour the contents into a beaker. Adjust the solution under test and the control solution to a pH of 1.8 in the same manner as before, and return the solutions to their respective separators. Add 5.0 mL of Diluted Dithizone Extraction Solution, shake vigorously, and allow the layers to separate. Using Diluted Dithizone Extraction Solution as a color blank, compare the colors developed in the chloroform layers of the solution under test and the control solution: the color developed by the solution under test is not more intense than that developed by the control solution (3 µg per g).Assay【试验】—Transfer【移取】500 mg of Ferrous Fumarate【富马酸亚铁】, accurately weighed【准确称重】, to a 500-mL conical flask【锥形瓶】, and add 25 mL of dilute hydrochloric acid (2 in 5)【(2→5)的稀盐酸】.Heat to boiling【加热至沸】, and add a solution of 5.6 g of stannous chloride【氯化亚锡】in 50 mL of dilute hydrochloric acid (3 in 10) 【(3→10)稀盐酸】dropwise【逐滴】until the yellow color disappears【黄色消失】, then add 2 drops in excess【过量2滴】.Cool the solution【溶液冷却】in an ice bath【冰浴】to room temperature【室温】, add 10 mL of mercuric chloride solution (1 in 20)【(1→20)氯化汞溶液】, and allow to stand for 5 minutes 【静置5分钟】.Add 200 mL of water, 25 mL of dilute sulfuric acid (1 in 2)【(1→2)稀盐酸】, and 4 mL of phosphoric acid【磷酸】, then add 2 drops of orthophenanthroline TS【邻菲罗啉(邻二氮菲)试液，TS=Test Solution 试液】, and titrate with 0.1 N ceric sulfate VS【用0.1N(0.1mol/L)硫酸铈标准滴定液滴定，VS=Volumetric Solution 滴定液】.Perform a blank determination【空白测定】, and make any necessary correction【做必要的修正】. Each mL of 0.1 N ceric sulfate is equivalent to【等于】16.99 mg of C4H2FeO4.Comments by Peng·L 2014年9月19日。
USP重金属检查法
标准操作规程Standard Operating Procedure1.简述1．1重金属是指在规定实验条件下能与硫代乙酰胺或硫化钠作用显色的金属盐类杂质。
1．2硫化钠或硫代乙酰胺在弱酸性条件下水解产生硫化氢，与供试品中重金属在规定实验条件下所显颜色，与一定量的标准铅溶液在同样操作条件下所显的颜色比较。
1．3由于实验条件不同。
分为三种检查方法：第一法适用于在规定条件下能生成澄清无色溶液的供试品。
第二法适用于在规定条件下不能生成澄清无色溶液的供试品；第三法适用于那些不能用一法和二法的样品。
2 仪器2．1仪器设备50ml纳氏比色管2．2试剂和溶液a)硫代乙酰胺试液: 称取硫代乙酰胺4g加水溶解，用水稀释至100ml，摇匀。
b)甘油基准试液: 称取200g甘油加水至总重量为235g,然后加氢氧化钠溶液和的水。
临用前用硫代乙酰胺试液和1ml甘油基准试液混合,在沸水浴中加热20秒钟,立即使用.c)硝酸铅贮备液：称取硝酸铅0.1598g，置1000ml量瓶中，加硝酸1ml与水100ml溶解后，用水稀释至1000ml 摇匀，作为贮备液。
标准铅溶液临用前，精密量取贮备液10ml，置100ml量瓶中，加水稀释至刻SOP-QM 402-14 页号 Page：2/4度，摇匀，即得（每1ml相当于10g的Pb）。
d）醋酸盐缓冲液: 取醋酸铵25.0g，加水25ml溶解后，加盐酸,如有必要用6N氨水或6N盐酸调PH至，用水稀释至100ml，摇匀。
第一法：标准溶液的制备取50ml比色管，用移液管移取标准铅溶液(20 gPb),用水稀释到25ml，用1N醋酸或6N氨水溶液调PH至~之间，用窄范围的精密pH试纸作指示，然后加水稀释至40ml，摇匀。
供试品溶液的制备另取一支50ml比色管，加入按该品种项下规定的方法制成的供试液25ml；或加入供试品的量以g计, 按公式1000L计算,其中L为重金属的限度(%)，用该品种项下规定的酸的体积溶解，加水溶解并稀释至25ml，供试品溶液用1N醋酸或6N氨水溶液调PH至～之间，用窄范围的精密pH试纸作指示,然后加水稀释至40ml，摇匀。
美国药典USP32-重金属测试
<231> 重金属本试验系在规定的试验条件下，金属离子与硫化物离子反应显色，通过制备的标准铅溶液目视比较测定，以确证供试品中重金属杂质含量不超过各论项下规定的限度（以供试品中铅的百分比表示，以重量计）。
（见分光光度法和光散射项下测定法目视比较法<851>）[ 注意:对本试验有响应的典型物质有铅、汞、铋、砷、锑、锡、镉、银、铜和钼等]。
除各论另有规定外，按第一法测定重金属。
第一法适用于在规定试验条件下，能产生澄清、无色溶液的物质。
第二法适用于在第一法规定试验条件下不能产生澄清、无色溶液的物质，或者适用于由于性质复杂，易干扰硫化物离子与金属离子形成沉淀的物质，或者是不易挥发的和易挥发的油类物质。
第三法为湿消化法，仅用于第一法、第二法都不适合的情况。
特殊试剂特殊试剂特殊试剂特殊试剂硝酸铅贮备液—取硝酸铅159.8mg，溶于100ml水中，加1ml硝酸，用水稀释至1000ml。
制备和贮存本溶液的玻璃容器应不含可溶性铅。
标准铅溶液—使用当天，取硝酸铅贮备液10.0ml，用水稀释至100.0ml。
每1ml的标准铅溶液含相当于10µg的铅。
按每克供试品取100µl标准铅溶液制备的对照溶液，相当于供试品含百万分之一的铅。
方法方法方法方法IIII pH3.5醋酸盐缓冲液—取醋酸铵25.0g溶于25ml水中，加6N盐酸液38.0ml，必要时，用6N氢氧化铵液或6N盐酸液调节pH至3.5，用水稀释至100ml，混匀。
标准溶液准备—精密量取标准铅溶液2ml，（相当于20µg的Pb），置50ml比色管中，加水稀释至25ml，以精密pH试纸作为外指示剂，用1N醋酸液或6N 氢氧化铵液调节pH至3.0~4.0，用水稀释至40ml，混匀。
供试品溶液制备—取各论项下规定的供试品溶液25ml，置50ml比色管中，或用各论项下规定用量的酸溶解样品，再用水稀释至25ml，供试品以g计，按下式计算： 2.0/（1000L）式中L是重金属限度（%）。
美国药典USP32测试溶液配制
TEST SOLUTIONS (TS)Certain of the following test solutions are intended for use as acid-base indicators in volumetric analyses. Such solutions should be so adjusted that when 0.15 mL of the indicator solution is added to 25 mL of carbondioxide-free water, 0.25 mL of 0.02 N acid or alkali, respectively, will produce the characteristic color change. Similar solutions are intended for use in pH measurement. Where no special directions for their preparation are given, the same solution is suitable for both purposes.Where it is directed that a volumetric solution be used as the test solution, standardization of the solution used as TS is not required.In general, the directive to prepare a solution ―fresh‖ indicates that the solution is of limited stability and must be prepared on the day of use.For the preparation of Test Solutions, use reagents of the quality described under Reagents.Acetaldehyde TS —Mix 4 mL of acetaldehyde, 3 mL of alcohol, and 1 mL of water. Prepare this solution fresh.Acetate Buffer TS —Dissolve 320 g of ammonium acetate in 500 mL of water, add 5 mL of glacial acetic acid, dilute with water to 1000.0 mL, and mix. This solution has a pH between 5.9 and 6.0.Acetic Acid, Glacial, TS —Determine the water content of a specimen of glacial acetic acid by the Titrimetric Method (see Water Determination 921). If the acid contains more than 0.05% of water, add a few mL of acetic anhydride, mix, allow to stand overnight, and again determine the water content. If the acid contains less than 0.02% of water, add sufficient water to make the final concentration between 0.02% and 0.05%, mix, allow to stand overnight, and again determine the water content. Repeat the adjustment withacetic anhydride or water, as necessary, until the resulting solution shows a water content between 0.02% and 0.05%.Acetic Acid, Strong, TS —Add 300.0 mL of glacial acetic acid, and dilute with water to 1000 mL. This solution contains about 30% (v/v) of CH3COOH and has a concentration of about 5 N.Acetic Acid–Ammonium Acetate Buffer TS —Dissolve 77.1 g of ammonium acetate in water, add 57 mL of glacial acetic acid, and dilute with water to 1000 mL.Acetone, Buffered, TS —Dissolve 8.15 g of sodium acetate and 42 g of sodium chloride in about 100 mL of water, and add 68 mL of 0.1 N hydrochloric acid and 150 mL of acetone. Mix, and dilute with water to 500 mL.Acid Ferric Chloride TS —Mix 60 mL of glacial acetic acid with 5 mL of sulfuric acid, add 1 mL of ferric chloride TS, mix, and cool.Acid Ferrous Sulfate TS —See Ferrous Sulfate, Acid, TS.Acid Stannous Chloride TS —See Stannous Chloride, Acid, TS.Acid Stannous Chloride TS, Stronger —See Stannous Chloride, Acid, TS.Albumen TS —Carefully separate the white from the yolk of a strictly fresh hen's egg. Shake the white with 100 mL of water until mixed and all but the chalaza has undergone solution; then filter. Prepare the solution fresh.Alcohol–Phenol TS —Dissolve 780 mg of phenol in alcohol to make 100 mL.Alcoholic Ammonia TS —See Ammonia TS, Alcoholic.Alcoholic Mercuric Bromide TS —See Mercuric Bromide TS, Alcoholic.Alcoholic Potassium Hydroxide TS —See Potassium Hydroxide TS, Alcoholic.Alkaline Cupric Citrate TS —See Cupric Citrate TS, Alkaline.Alkaline Cupric Citrate TS 2 —See Cupric Citrate TS 2, Alkaline.Alkaline Cupric Iodide TS —See Cupric Iodide TS, Alkaline.Alkaline Cupric Tartrate TS (Fehling's Solution)—See Cupric Tartrate TS, Alkaline.Alkaline Mercuric–Potassium Iodide TS —See Mercuric–Potassium Iodide TS, Alkaline.Alkaline Picrate TS —See Picrate TS, Alkaline.Alkaline Sodium Hydrosulfite TS —See Sodium Hydrosulfite TS, Alkaline. Amaranth TS —Dissolve 20 mg of amaranth in 10 mL of water.Aminonaphtholsulfonic Acid TS —Accurately weigh 5 g of sodium sulfite, 94.3 g of sodium bisulfite, and 700 mg of 1,2,4-aminonaphtholsulfonic acid, and mix. Prepare aminonaphtholsulfonic acid TS fresh on the day of use by dissolving 1.5 g of the dry mixture in 10 mL of water.Ammonia–Ammonium Chloride Buffer TS —Dissolve 67.5 g of ammonium chloride in water, add 570 mL of ammonium hydroxide, and dilute with water to 1000 mL.Ammonia–Cyanide TS —Dissolve 2 g of potassium cyanide in 15 mL of ammonium hydroxide, and dilute with water to 100 mL.Ammonia TS —It contains between 9.5% and 10.5% of NH3. Prepare by diluting 350 mL of Ammonia Water, Stronger (see in the section, Reagents) with water to make 1000 mL.Ammonia TS, Alcoholic —A solution of ammonia gas in alcohol. Clear, colorless liquid having a strong odor of ammonia. Specific gravity: about 0.80. It contains between 9% and 11% of NH3. Store it in alkali-resistant containers, in a cold place.Ammonia TS, Stronger —Use Ammonia Water, Stronger (see in the section Reagents).Ammoniacal Potassium Ferricyanide TS —Dissolve 2 g of potassium ferricyanide in 75 mL of water, add 25 mL of ammonium hydroxide, and mix.Ammoniated Cupric Oxide TS —See Cupric Oxide, Ammoniated, TS.Ammonium Acetate TS —Dissolve 10 g of ammonium acetate in water to make 100 mL.Ammonium Carbonate TS —Dissolve 20 g of ammonium carbonate and 20 mL of ammonia TS in water to make 100 mL.Ammonium Chloride TS —Dissolve 10.5 g of ammonium chloride in water to make 100 mL.Ammonium Chloride–Ammonium Hydroxide TS —Mix equal volumes of water and ammonium hydroxide, and saturate with ammonium chloride.Ammonium Molybdate TS —Dissolve 6.5 g of finely powdered molybdic acid in a mixture of 14 mL of water and 14.5 mL of ammonium hydroxide. Cool the solution, and add it slowly, with stirring, to a well-cooled mixture of 32 mL of nitric acid and 40 mL of water. Allow to stand for 48 hours, and filter through a fine-porosity, sintered-glass crucible. This solution deteriorates upon standing and is unsuitable for use if, upon the addition of 2 mL of dibasic sodium phosphate TS to 5 mL of the solution, an abundant yellow precipitate does not form at once or after slight warming. Store it in the dark. If a precipitate forms during storage, use only the clear supernatant.Ammonium Oxalate TS —Dissolve 3.5 g of ammonium oxalate in water to make 100 mL.Ammonium Phosphate, Dibasic, TS (Ammonium Phosphate TS)— Dissolve 13 g of dibasic ammonium phosphate in water to make 100 mL.Ammonium Polysulfide TS —Yellow liquid, made by saturating ammonium sulfide TS with sulfur.Ammonium Pyrrolidinedithiocarbamate, Saturated, TS —Add about 10 g of ammonium pyrrolidinedithiocarbamate to a 1000-mL volumetric flask, and dilute with water to volume.Ammonium Reineckate TS —Shake about 500 mg of ammonium reineckate with 20 mL of water frequently during 1 hour, and filter. Use within 2 days.Ammonium Sulfide TS —Saturate ammonia TS with hydrogen sulfide by bubbling hydrogen sulfide gas through the solution for 1 minute. This solution must be freshly prepared. The solution is not rendered turbid either by magnesium sulfate TS or by calcium chloride TS (carbonate). This solution is unstable for use if an abundant precipitate of sulfur is present.Residue on ignition: not more than 0.05%.Ammonium Thiocyanate TS —Dissolve 8 g of ammonium thiocyanate in water to make 100 mL.Ammonium Vanadate TS —Dissolve 2.5 g of ammonium vanadate in 500 mL of boiling water, cool, and add 20 mL of nitric acid. Mix, cool, and add water to make 1 L. Store in polyethylene containers.Anthrone TS —Within 12 hours of use, rapidly dissolve 35 mg of anthrone in a hot mixture of 35 mL of water and 65 mL of sulfuric acid. Immediately cool in an ice bath to room temperature, and filter through glass wool. Allow the solution to stand at room temperature for 30 minutes before use.Antimony Trichloride TS —Dissolve 20 g of antimony trichloride in chloroform to make 100 mL. Filter if necessary.Barium Chloride TS —Dissolve 12 g of barium chloride in water to make 100 mL.Barium Hydroxide TS —A saturated solution of barium hydroxide in recently boiled water. Prepare the solution fresh.Barium Nitrate TS —Dissolve 6.5 g of barium nitrate in water to make 100 mL.Betanaphthol TS —See 2-Naphthol TS.Biuret Reagent TS —Dissolve 1.5 g of cupric sulfate and 6.0 g of potassium sodium tartrate in 500 mL of water in a 1000-mL volumetric flask. Add 300 mL of carbonate-free sodium hydroxide solution (1 in 10), dilute withcarbonate-free sodium hydroxide solution (1 in 10) to 1000 mL, and mix.Blue Tetrazolium TS —Dissolve 500 mg of blue tetrazolium in alcohol to make 100 mL.Brilliant Blue G TS —Transfer 25 mg of brilliant blue G to a 100-mL volumetric flask, add 12.5 mL of alcohol and 25 mL of phosphoric acid, dilute with water to volume, and mix.Bromine TS (Bromine Water)— A saturated solution of bromine, prepared by agitating 2 to 3 mL of bromine with 100 mL of cold water in a glass-stoppered bottle, the stopper of which should be lubricated with petrolatum. Store it in a cold place, protected from light.Bromine–Sodium Acetate TS —Dissolve 100 g of sodium acetate in 1000 mL of glacial acetic acid, add 50 mL of bromine, and mix.p-Bromoaniline TS —Add 8 g of p-bromoaniline to a mixture of 380 mL ofthiourea-saturated glacial acetic acid, 10 mL of sodium chloride solution (1 in 5), 5 mL of oxalic acid solution (1 in 20), and 5 mL of dibasic sodium phosphate solution (1 in 10) in a low-actinic glass bottle. Mix, and allow to stand overnight before using. Protect from light, and use within 7 days.Bromocresol Blue TS —Use Bromocresol Green TS.Bromocresol Green TS —Dissolve 50 mg of bromocresol green in 100 mL of alcohol, and filter if necessary.Bromocresol Green–Methyl Red TS —Dissolve 0.15 g of bromocresol green and 0.1 g of methyl red in 180 mL of alcohol, and dilute with water to 200 mL.Bromocresol Purple TS —Dissolve 250 mg of bromocresol purple in 20 mL of 0.05 N sodium hydroxide, and dilute with water to 250 mL.Bromophenol Blue TS —Dissolve 100 mg of bromophenol blue in 100 mL of diluted alcohol, and filter if necessary.Bromothymol Blue TS —Dissolve 100 mg of bromothymol blue in 100 mL of diluted alcohol, and filter if necessary.Buffered Acetone TS —See Acetone, Buffered, TS.Calcium Chloride TS —Dissolve 7.5 g of calcium chloride in water to make 100 mL.Calcium Hydroxide TS —Use Calcium Hydroxide Topical Solution (USP monograph).Calcium Sulfate TS —A saturated solution of calcium sulfate in water.Ceric Ammonium Nitrate TS —Dissolve 6.25 g of ceric ammonium nitrate in 10 mL of 0.25 N nitric acid. Use within 3 days.Chloral Hydrate TS —Dissolve 50 g of chloral hydrate in a mixture of 15 mL of water and 10 mL of glycerin.Chlorine TS (Chlorine Water)— A saturated solution of chlorine in water. Place the solution in small, completely filled, light-resistant containers. Chlorine TS, even when kept from light and air, is apt to deteriorate. Store it in a cold, dark place. For full strength, prepare this solution fresh.Chromotropic Acid TS —Dissolve 50 mg of chromotropic acid or its disodium salt in 100 mL of 75% sulfuric acid, which may be made by cautiously adding 75 mL of sulfuric acid to 33.3 mL of water.Cobalt–Uranyl Acetate TS —Dissolve, with warming, 40 g of uranyl acetate in a mixture of 30 g of glacial acetic acid and sufficient water to make 500 mL. Similarly, prepare a solution containing 200 g of cobaltous acetate in a mixture of 30 g of glacial acetic acid and sufficient water to make 500 mL. Mix the two solutions while still warm, and cool to 20. Maintain the temperature at 20for about 2 hours to separate the excess salts from solution, and then pass through a dry filter.Cobaltous Chloride TS —Dissolve 2 g of cobaltous chloride in 1 mL of hydrochloric acid and sufficient water to make 100 mL.Congo Red TS —Dissolve 500 mg of congo red in a mixture of 10 mL of alcohol and 90 mL of water.m-Cresol Purple TS —Dissolve 0.10 g of metacresol purple in 13 mL of 0.01 N sodium hydroxide, dilute with water to 100 mL, and mix.Cresol Red TS —Triturate 100 mg of cresol red in a mortar with 26.2 mL of 0.01 N sodium hydroxide until solution is complete, then dilute the solution with water to 250 mL.Cresol Red–Thymol Blue TS —Add 15 mL of thymol blue TS to 5 mL of cresol red TS, and mix.Crystal Violet TS —Dissolve 100 mg of crystal violet in 10 mL of glacial acetic acid.Cupric Acetate TS —Dissolve 100 mg of cupric acetate in about 5 mL of water to which a few drops of acetic acid have been added. Dilute to 100 mL, and filter, if necessary.Cupric Acetate TS, Stronger (Barfoed's Reagent)— Dissolve 13.3 g of cupric acetate in a mixture of 195 mL of water and 5 mL of acetic acid.Cupric-Ammonium Sulfate TS —To cupric sulfate TS add ammonia TS, dropwise, until the precipitate initially formed is nearly but not completely dissolved. Allow to settle, and decant the clear solution. Prepare this solution fresh.Cupric Citrate TS —Dissolve 25 g of cupric sulfate, 50 g of citric acid, and 144 g of anhydrous sodium carbonate in water, and dilute with water to 1000 mL.Cupric Citrate TS, Alkaline —With the aid of heat, dissolve 173 g of dihydrated sodium citrate and 117 g of monohydrated sodium carbonate in about 700 mL of water, and filter through paper, if necessary, to obtain a clear solution. In a separate container dissolve 17.3 g of cupric sulfate in about 100 mL of water, and slowly add this solution, with constant stirring, to the first solution. Cool the mixture, add water to make 1000 mL, and mix.Cupric Citrate TS 2, Alkaline —With the aid of heat, dissolve about 173 g of sodium citrate dihydrate and 117 g of sodium carbonate monohydrate in about 700 mL of water, and filter. In a second flask, dissolve about 27.06 g of cupric sulfate (Cu2O4·5H2O) in about 100 mL of water. Slowly combine the two solutions while stirring, and dilute with water to 1000 mL.Cupric Iodide TS, Alkaline —Dissolve 7.5 g of cupric sulfate (CuSO4·5H2O) in about 100 mL of water. In a separate container dissolve 25 g of anhydrous sodium carbonate, 20 g of sodium bicarbonate, and 25 g of potassium sodium tartrate in about 600 mL of water. With constant stirring, add the cupric sulfate solution to the bottom of the alkaline tartrate solution by means of a funnel that touches the bottom of the container. Add 1.5 g of potassium iodide, 200 g of anhydrous sodium sulfate, 50 to 150 mL of 0.02 M potassium iodate, and sufficient water to make 1000 mL.Cupric Oxide, Ammoniated, TS (Schweitzer's Reagent)—Dissolve 10 g of cupric sulfate in 100 mL of water, add sufficient sodium hydroxide solution (1 in 5) to precipitate the copper hydroxide, collect the latter on a filter, and wash free from sulfate with cold water. Dissolve the precipitate, which must be keptwet during the entire process, in the minimum quantity of ammonia TS necessary for complete solution.Cupric Sulfate TS —Dissolve 12.5 g of cupric sulfate in water to make 100 mL.Cupric Tartrate TS, Alkaline (Fehling's Solution)—The Copper Solution (A)— Dissolve 34.66 g of carefully selected, small crystals of cupric sulfate, showing no trace of efflorescence of adhering moisture, in water to make 500 mL. Store this solution in small, tight containers.The Alkaline Tartrate Solution (B)— Dissolve 173 g of crystallized potassium sodium tartrate and 50 g of sodium hydroxide in water to make 500 mL. Store this solution in small, alkali-resistant containers.For use, mix exactly equal volumes of Solutions A and B at the time required.Delafield's Hematoxylin TS —Prepare 400 mL of a saturated solution of ammonium alum (Solution A). Dissolve 4 g of hematoxylin in 25 mL of alcohol, mix it with Solution A, and allow it to stand for 4 days in a flask closed with a pledget of purified cotton and exposed to light and air (Solution B). Then filter Solution B, and add to it a Solution C consisting of a mixture of 100 mL of glycerin and 100 mL of methanol. Mix, and allow the mixture to stand in a warm place, exposed to light, for 6 weeks until it becomes dark-colored. Store in tightly stoppered bottles.For use in staining endocrine tissue, dilute this test solution with an equal volume of water.Denigès' Reagent —See Mercuric Sulfate TS.Diazobenzenesulfonic Acid TS —Place in a beaker 1.57 g of sulfanilic acid,previously dried at 105for 3 hours, add 80 mL of water and 10 mL of diluted hydrochloric acid, and warm on a steam bath until dissolved. Cool to 15 (some of the sulfanilic acid may separate but will dissolve later), and add slowly, with constant stirring, 6.5 mL of sodium nitrite solution (1 in 10). Then dilute with water to 100 mL.Dichlorofluorescein TS —Dissolve 100 mg of dichlorofluorescein in 60 mL of alcohol, add 2.5 mL of 0.1 N sodium hydroxide, mix, and dilute with water to 100 mL.2,7-Dihydroxynaphthalene TS —Dissolve 100 mg of2,7-dihydroxynaphthalene in 1000 mL of sulfuric acid, and allow the solution to stand until the yellow color disappears. If the solution is very dark, discard it and prepare a new solution from a different supply of sulfuric acid. This solution is stable for approximately 1 month if stored in a dark bottle.Diiodofluorescein TS —Dissolve 500 mg of diiodofluorescein in a mixture of 75 mL of alcohol and 30 mL of water.Diluted Lead Subacetate TS —See Lead Subacetate TS, Diluted.p-Dimethylaminobenzaldehyde TS —Dissolve 125 mg ofp-dimethylaminobenzaldehyde in a cooled mixture of 65 mL of sulfuric acid and 35 mL of water, and add 0.05 mL of ferric chloride TS. Use within 7 days.Dinitrophenylhydrazine TS —Carefully mix 10 mL of water and 10 mL of sulfuric acid, and cool. To the mixture, contained in a glass-stoppered flask, add 2 g of 2,4-dinitrophenylhydrazine, and shake until dissolved. To the solution add 35 mL of water, mix, cool, and filter.Diphenylamine TS —Dissolve 1.0 g of diphenylamine in 100 mL of sulfuric acid. The solution should be colorless.Diphenylcarbazone TS —Dissolve 1 g of crystalline diphenylcarbazone in 75 mL of alcohol, then add alcohol to make 100 mL. Store in a brown bottle.Dithizone TS —Dissolve 25.6 mg of dithizone in 100 mL of alcohol. Store in a cold place, and use within 2 months.Dragendorff's TS —Mix 850 mg of bismuth subnitrate with 40 mL of water and 10 mL of glacial acetic acid (Solution A). Dissolve 8 g of potassium iodide in 20 mL of water (Solution B). Mix equal portions of Solution A and Solution B to obtain a stock solution, which can be stored for several months in a dark bottle. Mix 10 mL of the stock solution with 20 mL of glacial acetic acid, and dilute with water to make 100 mL.Edetate Disodium TS —Dissolve 1 g of edetate disodium in 950 mL of water, add 50 mL of alcohol, and mix.Eosin Y TS (adsorption indicator)—Dissolve 50 mg of eosin Y in 10 mL of water.Eriochrome Black TS —Dissolve 200 mg of eriochrome black T and 2 g of hydroxylamine hydrochloride in methanol to make 50 mL.Eriochrome Cyanine TS —Dissolve 750 mg of eriochrome cyanine R in 200 mL of water, add 25 g of sodium chloride, 25 g of ammonium nitrate, and 2 mL of nitric acid, and dilute with water to 1000 mL.Fehling's Solution —See Cupric Tartrate TS, Alkaline.Ferric Ammonium Sulfate TS —Dissolve 8 g of ferric ammonium sulfate in water to make 100 mL.Ferric Chloride TS —Dissolve 9 g of ferric chloride in water to make 100 mL.Ferroin TS —Dissolve 0.7 g of ferrous sulfate and 1.76 g of o-phenanthroline monohydrochloride monohydrate in water, and dilute with water to 100 mL.Ferrous Sulfate TS —Dissolve 8 g of clear crystals of ferrous sulfate in about 100 mL of recently boiled and thoroughly cooled water. Prepare this solution fresh.Ferrous Sulfate, Acid, TS —Dissolve 7 g of ferrous sulfate crystals in 90 mL of recently boiled and thoroughly cooled water, and add sulfuric acid to make 100 mL. Prepare this solution immediately prior to use.Folin-Ciocalteu Phenol TS —Into a 1500-mL flask introduce 100 g of sodium tungstate, 25 g of sodium molybdate, 700 mL of water, 50 mL of phosphoric acid, and 100 mL of hydrochloric acid. Gently reflux the mixture for about 10 hours, and add 150 g of lithium sulfate, 50 mL of water, and a few drops of bromine. Boil the mixture, without the condenser, for about 15 minutes, or until the excess bromine is expelled. Cool, dilute with water to 1 L, and filter: the filtrate has no greenish tint. Before use, dilute 1 part of the filtrate with 1 part of water. When used for protein determination (i.e., Lowry assay), this reagent must be further diluted (1:5) with water. See Method 2 in Total Protein Assay under Biotechnology-Derived Articles—Total Protein Assay 1057.Formaldehyde TS —Use Formaldehyde Solution (see in the section Reagents).Fuchsin–Pyrogallol TS —Dissolve 100 mg of basic fuchsin in 50 mL of water that previously has been boiled for 15 minutes and allowed to cool slightly. Cool, add 2 mL of a saturated solution of sodium bisulfite, mix, and allow to stand for not less than 3 hours. Add 0.9 mL of hydrochloric acid, mix, and allow to stand overnight. Add 100 mg of pyrogallol, shake until solution is effected, and dilute with water to 100 mL. Store in an amber-colored glass bottle in a refrigerator.Fuchsin–Sulfurous Acid TS —Dissolve 200 mg of basic fuchsin in 120 mL of hot water, and allow the solution to cool. Add a solution of 2 g of anhydrous sodium sulfite in 20 mL of water, then add 2 mL of hydrochloric acid. Dilute the solution with water to 200 mL, and allow to stand for at least 1 hour. Prepare this solution fresh.Gastric Fluid, Simulated, TS —Dissolve 2.0 g of sodium chloride and 3.2 g of purified pepsin, that is derived from porcine stomach mucosa, with an activity of 800 to 2500 units per mg of protein, in 7.0 mL of hydrochloric acid and sufficient water to make 1000 mL. [note—Pepsin activity is described in the Food Chemicals Codex specifications under General Tests and Assays.] This test solution has a pH of about 1.2.Gelatin TS (for the assay of Corticotropin Injection)—Dissolve 340 g ofacid-treated precursor gelatin (Type A) in water to make 1000 mL. Heat the solution in an autoclave at 115for 30 minutes after the exhaust linetemperature has reached 115. Cool the solution, and add 10 g of phenol and 1000 mL of water. Store in tight containers in a refrigerator.Glacial Acetic Acid TS —See Acetic Acid, Glacial, TS.Glucose Oxidase–Chromogen TS —A solution containing, in each mL, 0.5µmol of 4-aminoantipyrine, 22.0 µmol of sodium p-hydroxybenzoate, not less than 7.0 units of glucose oxidase, and not less than 0.5 units of peroxidase, and buffered to a pH of 7.0 ± 0.1.Suitability —When used for determining glucose in Inulin, ascertain that no significant color results by reaction with fructose, and that a suitable absorbance-versus-concentration slope is obtained with glucose.[note—A suitable grade is available, as a concentrate, from Worthington Diagnostics, Division of Millipore Corp., .]Glycerin Base TS —To 200 g of glycerin add water to bring the total weight to 235 g. Add 140 mL of 1 N sodium hydroxide and 50 mL of water.Gold Chloride TS —Dissolve 1 g of gold chloride in 35 mL of water.Hydrogen Peroxide TS —Use Hydrogen Peroxide Topical Solution (USP monograph).Hydrogen Sulfide TS —A saturated solution of hydrogen sulfide, made by passing H2S into cold water. Store it in small, dark amber-colored bottles, filled nearly to the top. It is unsuitable unless it possesses a strong odor of H2S, and unless it produces at once a copious precipitate of sulfur when added to an equal volume of ferric chloride TS. Store in a cold, dark place.Hydroxylamine Hydrochloride TS —Dissolve 3.5 g of hydroxylamine hydrochloride in 95 mL of 60% alcohol, and add 0.5 mL of bromophenol blue solution (1 in 1000 of alcohol) and 0.5 N alcoholic potassium hydroxide until a greenish tint develops in the solution. Then add 60% alcohol to make 100 mL.8-Hydroxyquinoline TS —Dissolve 5 g of 8-hydroxyquinoline in alcohol to make 100 mL.Indigo Carmine TS (Sodium Indigotindisulfonate TS)— Dissolve a quantity of sodium indigotindisulfonate, equivalent to 180 mg of C16H8N2O2(SO3Na)2, in water to make 100 mL. Use within 60 days.Indophenol–Acetate TS (for the assay of Corticotropin Injection)—To 60 mL of standard dichlorophenol-indophenol solution (see in the section Volumetric Solutions) add water to make 250 mL. Add to the resulting solution an equal volume of sodium acetate solution freshly prepared by dissolving 13.66 g of anhydrous sodium acetate in water to make 500 mL and adjusting with 0.5 N acetic acid to a pH of 7. Store in a refrigerator, and use within 2 weeks.Intestinal Fluid, Simulated, TS —Dissolve 6.8 g of monobasic potassium phosphate in 250 mL of water, mix, and add 77 mL of 0.2 N sodium hydroxide and 500 mL of water. Add 10.0 g of pancreatin, mix, and adjust the resulting solution with either 0.2 N sodium hydroxide or 0.2 N hydrochloric acid to a pH of 6.8 ± 0.1. Dilute with water to 1000 mL.Iodine TS —Use 0.1 N Iodine (see in the section Volumetric Solutions).Iodine, Diluted TS— Transfer 10.0 mL of 0.1 N iodine VS to a 100-mL volumetric flask, dilute with water to volume, and mix.Iodine Monochloride TS —Dissolve 10 g of potassium iodide and 6.44 g of potassium iodate in 75 mL of water in a glass-stoppered container. Add 75 mL of hydrochloric acid and 5 mL of chloroform, and adjust to a faint iodine color (in the chloroform) by adding dilute potassium iodide or potassium iodate solution. If much iodine is liberated, use a stronger solution of potassium iodate than 0.01 M at first, making the final adjustment with the 0.01 M potassium iodate. Store in a dark place, and readjust to a faint iodine color as necessary.Iodine and Potassium Iodide TS 1 —Dissolve 500 mg of iodine and 1.5 g of potassium iodide in 25 mL of water.Iodine and Potassium Iodide TS 2 —Dissolve 12.7 g of iodine and 20 g of potassium iodide in water, and dilute with water to 1000.0 mL. To 10.0 mL of this solution, add 0.6 g of potassium iodide, and dilute with water to 100.0 mL. Prepare immediately before use.Iodobromide TS —Dissolve 20 g of iodine monobromide in glacial acetic acid to make 1000 mL. Store in glass containers, protected from light.Iodochloride TS —Dissolve 16.5 g of iodine monochloride in 1000 mL of glacial acetic acid.Iodoplatinate TS —Dissolve 300 mg of platinic chloride in 97 mL of water. Immediately prior to use, add 3.5 mL of potassium iodide TS, and mix.Iron–Phenol TS (Iron-Kober Reagent)— Dissolve 1.054 g of ferrous ammonium sulfate in 20 mL of water, and add 1 mL of sulfuric acid and 1 mL of 30 percent hydrogen peroxide. Mix, heat until effervescence ceases, and dilutewith water to 50 mL. To 3 volumes of this solution contained in a volumetric flask add sulfuric acid, with cooling, to make 100 volumes. Purify phenol by distillation, discarding the first 10% and the last 5%, collecting the distillate, with exclusion of moisture, in a dry, tared glass-stoppered flask of about twice the volume of the phenol. Solidify the phenol in an ice bath, breaking the top crust with a glass rod to ensure complete crystallization. Weigh the flask and its contents, add to the phenol 1.13 times its weight of the iron–sulfuric acid solution prepared as directed, insert the stopper in the flask, and allow to stand, without cooling but with occasional mixing, until the phenol is liquefied. Shake the mixture vigorously until mixed, allow to stand in the dark for 16 to 24 hours, and again weigh the flask and its contents. To the mixture add 23.5% of its weight of a solution of 100 volumes of sulfuric acid in 110 volumes of water, mix, transfer to dry glass-stoppered bottles, and store in the dark, protected from atmospheric moisture. Use within 6 months. Dispense the reagent from a small-bore buret, arranged to exclude moisture, capable of delivering 1 mL in 30 seconds or less, and having no lubricant, other than reagent, on its stopcock. Wipe the buret tip with tissue before each addition.Iron Salicylate TS —Dissolve 500 mg of ferric ammonium sulfate in 250 mL of water containing 10 mL of diluted sulfuric acid, and add water to make 500 mL. To 100 mL of the resulting solution add 50 mL of a 1.15% solution of sodium salicylate, 20 mL of diluted acetic acid, and 80 mL of a 13.6% solution of sodium acetate, then add water to make 500 mL. Store in a well-closed container. Protect from light. Use within 2 weeks.Lead Acetate TS —Dissolve 9.5 g of clear, transparent crystals of lead acetate in recently boiled water to make 100 mL. Store in well-stoppered bottles.。
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<231>重金属本试验系在规定的试验条件下，金属离子与硫化物离子反应显色，通过制备的标准铅溶液目视比较测定，以确证供试品中重金属杂质含量不超过各论项下规定的限度（以供试品中铅的百分比表示，以重量计）。
（见分光光度法和光散射项下测定法目视比较法<851>）[注意:对本试验有响应的典型物质有铅、汞、铋、砷、锑、锡、镉、银、铜和钼等]。
除各论另有规定外，按第一法测定重金属。
第一法适用于在规定试验条件下，能产生澄清、无色溶液的物质。
第二法适用于在第一法规定试验条件下不能产生澄清、无色溶液的物质，或者适用于由于性质复杂，易干扰硫化物离子与金属离子形成沉淀的物质，或者是不易挥发的和易挥发的油类物质。
第三法为湿消化法，仅用于第一法、第二法都不适合的情况。
特殊试剂特殊试剂硝酸铅贮备液—取硝酸铅159.8mg，溶于100ml水中，加1ml硝酸，用水稀释至1000ml。
制备和贮存本溶液的玻璃容器应不含可溶性铅。
标准铅溶液—使用当天，取硝酸铅贮备液10.0ml，用水稀释至100.0ml。
每1ml的标准铅溶液含相当于10µg的铅。
按每克供试品取100µl标准铅溶液制备的对照溶液，相当于供试品含百万分之一的铅。
方法方法II pH3.5醋酸盐缓冲液—取醋酸铵25.0g溶于25ml水中，加6N盐酸液38.0ml，必要时，用6N氢氧化铵液或6N盐酸液调节pH至3.5，用水稀释至100ml，混匀。
标准溶液准备—精密量取标准铅溶液2ml，（相当于20µg的Pb），置50ml比色管中，加水稀释至25ml，以精密pH 试纸作为外指示剂，用1N醋酸液或6N氢氧化铵液调节pH至3.0~4.0，用水稀释至40ml，混匀。
供试品溶液制备—取各论项下规定的供试品溶液25ml，置50ml比色管中，或用各论项下规定用量的酸溶解样品，再用水稀释至25ml，供试品以g计，按下式计算：2.0/（1000L）式中L是重金属限度（%）。
以精密pH试纸作为外指示剂，用1N醋酸液或6N氢氧化铵液调节pH至3.0~4.0，用水稀释至40ml，摇匀。
对照溶液制备—取供试品溶液制备项下的溶液25ml，置50ml比色管中，加标准铅溶液2.0ml，以精密pH试纸作为外指示剂，用1N醋酸液或6N氢氧化铵液调节pH至3.0~4.0，用水稀释至40ml，摇匀。
测定法—在上述三试管中，分别加入pH3.5的醋酸盐缓冲液2ml，然后再加硫代乙酰胺—甘油试液1.2ml，用水稀释至50ml，混匀，放置2分钟，在白色平面∗自上向下观察：供试品溶液产生的颜色与标准品溶液产生的颜色相比，不得更深。
对照溶液产生的颜色比标准溶液深或相当。
[注意：如果对照溶液的颜色比标准溶液浅，用方法II代替方法I测定供试品]。
方法方法IIII pH3.5醋酸盐缓冲液—按方法I配制。
标准溶液准备—按方法I配制。
供试品溶液制备—供试品以g计，按下式计算：2.0/（1000L）式中L是重金属限度（%）。
取供试品适量，称重，置适宜的坩埚中，加适量的硫酸使湿润，低温小心灼烧，直至全部炭化，（在炭化过程中坩埚不可盖严），加硝酸2ml和硫酸5滴至炭化物上，小心加热直到白烟不再逸出，置马富炉中500~600°灼烧，直至完全灰化，放冷，加6N盐酸液4ml，加盖，置蒸气浴上加热15分钟，去盖，在蒸汽浴上慢慢蒸发至干，用1滴盐酸湿润残渣，加热水10ml，蒸煮2分钟，滴加6N氢氧化铵液，直到溶液对石蕊试纸呈碱性，用水稀释至25ml，以精密pH试纸作为外指示剂，用1N醋酸液调节pH至3.0~4.0，必要时，滤过，用10ml水洗涤坩埚和滤器，合并滤液和洗液，置50ml比色管中，用水稀释至40ml，摇匀。
测定法—在上述二试管中，分别加入pH3.5的醋酸盐缓冲液2ml，然后再加硫代乙酰胺—甘油试液1.2ml，用水稀释至50ml，混匀，放置2分钟，在白色平面∗自上向下观察：供试品溶液产生的颜色与标准品溶液产生的颜色相比，不得更深。
方法方法IIIIII pH3.5醋酸盐缓冲液——按方法I所示的方法配制。
标准溶液的制备——取硫酸8mL和硝酸10mL的混合液，置洁净干燥的100mL凯氏烧瓶中，再加硝酸适量，加入量与供试品溶液中加入的硝酸量相当。
加热使产生浓的白烟，冷却，小心加水10mL，若处理供试品需用过氧化氢，则加30%过氧化氢适量，加入量相当于供试品中消耗的过氧化氢量。
缓缓煮沸至产生浓的白烟，再冷却，小心地加水5mL，混匀，缓缓煮沸至产生浓的白烟，浓缩至体积2~3mL，冷却，小心加水数毫升稀释，加标准铅溶液2.0mL（20µ的铅），混匀，移入50mL比色管中，用水洗涤烧瓶，洗液并入比色管中，并稀释至25mL，混匀。
供试品溶液的制备—-若供试品为固体——按各论中的规定称取供试品适量，置洁净干燥的100mL凯氏烧瓶中[注意——若反应泡沫过多，可用300mL的烧瓶]，夹住烧瓶使成45°角，加入硫酸8mL和硝酸10mL的混合液适量，其量应足以使样品完全湿润，缓缓加热，至反应开始后停止加热，待反应平息，再分数次加入上述剩余的酸混合液，每次加酸后再加热，直至18mL酸混合液全部加完。
继续加热至微沸，直至溶液变黑，冷却，加硝酸2mL，再加热至溶液变黑。
继续加热，再加硝酸，直至溶液不再变黑，然后加强热使产生浓的白烟，冷却，小心地加入水5mL，缓缓加热至产生浓的白烟，继续加热直至体积仅剩数毫升，冷却，小心地加水5mL，观察溶液颜色，若呈黄色，则小心地加入30%的过氧化氢1mL，再蒸发至产生浓的白烟且体积仅剩2~3mL，若溶液仍呈黄色，可重复加水5mL及过氧化氢处理。
冷却，小心地加水数毫升稀释，并洗入50mL比色管中，注意合并洗液后的体积不得超过25mL。
若供试品为液体——取各论中规定量的供试品，置一洁净干燥的100mL凯氏烧瓶中[注——若反应泡沫过多，可用300mL烧瓶]，夹住烧瓶使成45°角，小心地加入硫酸8mL与硝酸10mL的混合液数毫升，缓缓温热至反应开始，待反应渐止，按固体样品项下自“分数次加入上述相同的酸混合液”起，同法处理。
检查法——供试品溶液及标准品溶液制备均按以下方法处理：用氢氧化铵调节pH值为3.0~4.0，用精密pH试纸为外指示剂（当接近规定的pH值时可用稀氨溶液），然后用水稀释至40mL，混匀。
每支比色管中加入pH3.5的醋酸盐缓冲液2mL，然后加硫乙酰氨——甘油碱性试液1.2mL，再加水稀释至50mL，混匀，静置2分钟，置白色平面上自上向下观察，供试品溶液的颜色与标准品溶液的颜色相比，不得更深the color of the solution from the Test Preparation is not darker than that of the solution from the Standard Preparation, and the color of the solution from the Monitor Preparation is equal to or darker than that of the solution from the Standard Preparation.[NOTE—If the color of the Monitor Preparation is lighter than that of the Standard Preparation, use Method II instead of Method I for the substance being tested.]Method II NOTE—This method does not recover mercury.pH3.5 Acetate Buffer—Prepare as directed under Method I.Standard Preparation—Pipet 4 mL of the Standard Lead Solution into a suitable test tube,and add 10mL of6N hydrochloric acid.Test Preparation—Use a quantity, in g, of the substance to be tested as calculated by the formula:the color of the Test Preparation is not darker than that of the Standard Preparation, and the color of the Monitor Preparation is equal to or darker than that of the Standard Preparation.见方法Ia项下原理部分给出的信息。
在残留滴定中，额外的试剂被加入到供试样品中，为反应的完成留下了充分的时间，并且将未消耗掉的试剂与水和某种溶剂（例如，甲醇）的标准溶液一起滴定。
残留滴定程序通常是可行的，并避免了可能在直接滴定该物质过程中遇到的困难，这些物质中被束缚水分释放得很缓慢。
Apparatus, Reagent, and Test Preparation—Use Method Ia.仪器、试剂、供试配制液：同方法Ia。
Standardization of Water Solution for Residual Titration—Prepare a Water Solution by diluting 2 mL of water with methanol or other suitable solvent to 1000 mL. Standardize this solution by titrating 25.0 mL with the Reagent, previously standardized as directed under Standardization of the Reagent. Calculate the water content, in mg per mL, of the Water Solution taken by the formula:用于残留滴定的水溶液的标准化：以甲醇或其他适当溶剂将2mL水稀释至1000mL，以配制水溶液。
使用此前已经按照试剂的标准化项下规定进行过标准化的试剂，对25mL此溶液进行滴定，从而对其进行标准化。
按照下面的公式，计算此水溶液中的水分含量（单位mg/mL）：V′F/25,步骤：当具体各论中规定用方法Ib测定水分含量时，将35至40mL适当溶剂转移至该滴定容器，并用试剂滴定至测电法或视觉观察的终点并加入精确称量的额外试剂。