畜禽肉制品中5种核苷酸的测定编制说明

《畜禽肉制品中5种核苷酸的测定》编制说明研究背景我国是一个畜禽肉制品生产消费大国，随着我国国民经济发展，消费者从关心食品安全到注重食品品质的转变。其中，核苷酸作为肉制品呈味物质已被广泛认同，游离核苷酸是已经成为评判畜禽肉制品品质及滋味的重要指标。畜禽肉制品中，对肉制品味道产生影响主要作用的核苷酸包括腺苷酸（Adenosine5'-monophosphate，AMP）、肌苷酸（Inosinemonphosphate，IMP）、鸟苷酸（Guanosine monophosphate，GMP）、肌苷（Hypoxanthineriboside，HxR）、次黄嘌呤（Hypoxanthine，Hx）5种，具体结构如图1所示。IMP、GMP、AMP本身就具有鲜味，并且会与谷氨酸钠之间呈现强烈鲜味的相乘作用，Hx具有苦味，会给肉的整体风味带来负面作用，5种核苷酸之间组成和相互作用，形成畜禽肉制品的不同风味。食品在加工过程中，细胞结构被破坏，DNA暴露，在酶及物理条件作用下发生裂解，大部分三磷酸腺苷（Adenosine5'-Triphosphate，ATP）降解为AMP，进一步降解成IMP，IMP后续再次降解，产生HxR与Hx。在生产过程中，生产者根据产品工艺需要，也会人为添加核苷酸。在GB2760-2014中就规定IMP和GMP可在各类食品中按生产需要适量使用。HxHxHxHxHxRGMPIMPAMP图15种核苷酸的结构式HxRGMPIMPAMP目前，国际标准化组织（ISO）对于核苷酸的关注集中在婴儿配方奶粉中，已有BSISO20638《婴儿配方奶粉液相色谱法测定核苷酸》，但是还没有关于肉制品的核苷酸检验标准。在我国仅有添加剂标准GB1886.97-2015《食品安全国家标准食品添加剂5'-肌苷酸二钠》，没有肉制品中核苷酸测定方法的国家标准。在GB2760-2024中规定肌苷酸二钠和鸟苷酸二钠可在各类食品中按生产需要适量使用，但并没有对应检测手段，国家层面的标准更新滞后。近几年，一些企业和团体为了适应行业发展，也纷纷颁布团体标准，其中畜禽肉品质指标的团体标准TNAIA003-2020《肌肉中肌苷肌苷酸的测定高效液相色谱法》于2020年发布。本标准能够助力企业在产品研发时，对产品滋味的控制，促进企业产品升级，丰富产品品类。本标准针对肉制品基质，采用液相色谱法进行检测。工作情况任务由来、起草单位、起草人根据《工业和信息化部办公厅关于印发2023年第二批行业标准制修订和外文版项目计划的通知》（工信厅科〔2023〕42号），由全国肉禽蛋制品标准化技术委员会（SAC/TC399）负责《畜禽肉制品中5种核苷酸的测定》行业标准（计划编号为：2023-0950T-QB）的制定工作。中国肉类食品综合研究中心承担《禽肉制品中5种核苷酸的测定》的起草工作。主要起草人员为赵燕、郭文萍、赵文涛、任南、王真、纪晓萌。简要起草过程2023年8月，我单位接到国家标准修订任务后，对该标准的具体工作进行了认真研究，确定了总体工作方案，并组建了标准修订工作组，制定了实施计划。2023年8月-12月，成立标准编制小组。全面了解国内外有关核苷酸的检测方法以及标准制定等情况，查阅国内外相关参考文献,了解目前核苷酸检测的现状，对相关企业及个人开展调查，完成实施方案的制定。2024年1月-7月，实验研究。开展试验研究，确定核苷酸测定方法的具体技术指标。2024年8月1号，于中国肉类食品综合研究中心院内召开行业标准起草工作组会议，起草组介绍标准的相关内容，与会专家针对标准草案逐条讨论，提出意见、建议。根据专家提出的修改意见，起草组对标准文本进行修改。方法的起草原则在编制过程中，本起草组查阅了相关文献报道，参考了各行业部门制定的同类标准方法，注重标准的可操作性，完成了检测方法的研究工作，形成了方法草案和编制说明。本方法的主要参数、公式、性能要求等主要依据GB/T1.1-2009《标准化工作指导》、GB/T20001.4-2015《标准编写规则》的要求进行编写。并且按照GB/T27404-2008《实验室质量控制规范》对方法学进行了考察。标准主要技术内容本标准研制过程中，通过查阅大量国内外有关文献，全面掌握了IMP、GMP、AMP、HxR、Hx的化学结构和理化特性。在此基础上，反复多次进行试验，针对前处理方法、仪器条件、线性范围、灵敏度和精确度等进行探索，并根据本实验条件进行了多次试验，最终确定了该方法的可行性，完成了检测方法标准的研究工作。仪器条件的优化检测波长的确定参考有关文献，运用光电二级阵列管检测器，对5种核苷酸进行扫描得到光谱图2，发现HxR、IMP在248nm有最大吸收，GMP在254nm有最大吸收，AMP在259nm有最大吸收，Hx在250nm有最大吸收。将色谱图叠加得到图3，在254nm波长处，AMP、HxR光谱形成交叉点，Hx、IMP光谱形成交叉点，并且都具有较高的响应，同时GMP在此波长下响应值也较高，所以最终实验确定选用检测波长为254nm。在该波长下，5种核苷酸的响应均可以满足方法检出限（limitofdetection，LOD）和定量限（limitofquantitation，LOQ）的要求。图25种核苷酸的光谱图图3不同波长下5种核苷酸的响应值色谱条件的确定选择合适的色谱条件，保证5种核苷酸的色谱峰可以实现基线分离，是进行研究的第一步。在色谱柱和流动相选择上，比较C18柱、HilicPlus柱、SB-Aq柱3种色谱柱，同时选取水/甲醇、甲酸水/甲醇、磷酸二氢钾/甲醇、乙酸铵/甲醇的流动相体系，在等度洗脱条件下进行分离。通过查阅文献可知，在5种核苷酸中，GMP和IMP较为难分离。结果发现，无论在何种流动相条件下，HilicPlus柱、SB-Aq柱均无法将GMP和IMP分离，图4为HilicPlus柱所得的色谱图，图5为SB-Aq柱所得的色谱图。C18柱在磷酸二氢钾/甲醇流动相体系下，可以成功分离5种核苷酸。最终确定色谱柱为C18柱（250mm×4.6mm，5μm）；流动相：0.01mol/L磷酸二氢钾∶甲醇＝98∶2（V/V）；流速：1.0mL/min；检测波长：254nm；进样量：20μL。HxR、IMP、GMP、AMP、Hx（10μg/mL）标准溶液的色谱图见图6。IMPGMPIMPGMP图4IMPGMPIMPGMP图5SB-Aq柱所得的色谱图GMPGMPIMPHxAMPHxR图6C18柱所得的5种核苷酸标准溶液色谱图（10μg/mL）柱温的确定调节柱温箱的温度，观察不同柱温下色谱峰的出峰时间和分离度的变化。分别考察了柱温为25、30、35℃时的5种核苷酸的出峰情况，结果如图7所示。结果表明，5种核苷酸的出峰时间在25及30℃条件下，出峰时间变化较小；35℃条件下，5种核苷酸的出峰时间后移，并且峰面积变小；不同温度对于5种核苷酸的分离度影响较小。考虑到实验室通常的仪器配置及检测的环境温度，因此选择柱温为25℃。ABCA.25℃；B.30℃；C.35℃图7不同柱温下5种核苷酸的出峰情况前处理条件的优化提取溶剂的确定作为化合物分析的第1步，选择合适的提取溶剂，将目标物从样品基质中高效、快速地提取出来，直接影响方法的提取效率和后续的净化步骤。本实验选择5%高氯酸＋5%甲醇水溶液、5%高氯酸、5%三氯乙酸、100%乙腈作为提取溶剂，分别考察4种溶剂的提取效果。图8不同提取溶剂对加标回收率的影响(n=3)对比加标量为100mg/kg时的加标回收率。由图8可知，用100%乙腈溶液作提取溶剂时，5种核苷酸基本没有回收，说明核苷酸很难溶解于有机溶剂，从而影响提取效率。用5%三氯乙酸溶液作提取溶剂时，目标化合物的加标回收率在62%左右。用5%高氯酸溶液和5%高氯酸＋5%甲醇水溶液作提取溶剂时，目标化合物的加标回收率在76%左右，但甲醇的加入会使实验平行性变差，实验偏差过大。因此选用高氯酸溶液作为提取溶剂。进一步对提取液的浓度进行优化。选择不同体积分数的高氯酸（1%、5%、10%、15%），分别考察其提取效果。对比加标量为100mg/kg时的加标回收率，实验结果如图9。结果表明，当高氯酸的体积分数为1%时，回收率为50%左右；当高氯酸体积分数为15%，回收率为63%左右，但结果偏差较大；当高氯酸体积分数为5%、10%时，回收率约为74%。两者回收率没有明显差异，考虑到酸会对后续处理产生影响，因此选择体积分数较低的5%高氯酸溶液作为提取溶剂。图9不同浓度的高氯酸对加标回收率的影响(n=3)提取方式的确定畜禽肉制品中脂肪和蛋白质的含量都很高，会对化合物的提取会有一定影响。选择合适的提取方式，有助于减小基质对于化合物的干扰，减少净化难度。本实验考察常见的4种提取方式：室温超声30min、冰浴超声30min、常温振荡器振荡30min、添加陶瓷均质子涡旋30min。图10不同提取方式对加标回收率的影响(n=3)对比加标量为100mg/kg时的加标回收率，实验结果如图10。室温超声30min的回收率约为68%，冰浴超声30min的回收率约为71%，两者没有明显差异。常温振荡器振荡30min的回收率约为80%，添加陶瓷均质子涡旋30min的回收率约为77%，两者在回收率上无明显差异。但添加陶瓷均质子涡旋30min得到的提取溶液表面有较多油脂悬浮，会对后续操作产生干扰，对比照片见图11。因此在提取时选择常温振荡器振荡30min。图11添加陶瓷均质子涡旋30min（左）、常温振荡器振荡30min（右）得到的提取溶液的状态进一步对常温振荡器振荡过程的提取时间进行优化。选择不同提取时间（10、20、30、40min），分别考察其提取效果。对比加标量为100mg/kg时的加标回收率，实验结果如图12。结果表明，在提取10min后，回收约为50%，且偏差较大，说明还未提取完全。随着提取时间的延长，回收率逐渐升高，从30min到40min时，回收率逐渐稳定在80%左右。因此选择提取时间为30min。ABCDEA.AMP；B.IMP；C.GMP；D.HxR；E.Hx图12不同提取时间对加标回收率的影响(n=3)离心转速的选择在样品振荡提取后进行离心，可以将样品中不溶物沉降，实现提取液的固液分离，方便进行下一步操作。离心转速是该过程的核心参数。本实验考察6000、8000、12000、15000r/min的转速，观察其离心效果。实验结果如图13所示。在6000及8000r/min条件下，上清液仍旧有少量固体物质残留，上层溶液比较浑浊；在12000及15000r/min条件下，上清液明显变澄清，说明不溶物已经沉降完全，且12000及15000r/min的状态大致相同。因此，实验中选择离心转速为12000r/min。6000r/min6000r/min8000r/min12000r/min15000r/min图13不同离心机转速的沉降效果比较提取液最终pH的确定在提取过程中，需要用到大量的酸来保证游离核苷酸充分离子化，充分溶解。但提取液中酸会对后续操作产生干扰，也会影响色谱柱柱效，影响分析物保留时间和分离效果。因此，在定容之前需要对提取液的pH进行调整。本实验比较不同pH值提取溶剂（5.5、6.0、6.5、7.0）的提取效果。ABCDEA.AMP；B.IMP；C.GMP；D.HxR；E.Hx图14提取液最终pH对加标回收率的影响(n=3)对比加标量为100mg/kg时的加标回收率，实验结果如图14。提取溶液pH从5.5到6.5，回收率随pH的升高而升高，提取溶液pH从6.5到7.0，回收率随pH的升高而降低，在pH6.5时回收率最高。因此选择提取液pH为6.5。为了进一步锁定合适的pH范围，对pH进一步细化。本实验设置了几个不同的提取液pH（6.1、6.3、6.5、6.7、6.9），探究了其对回收率的影响。实验结果如图15。取溶液pH从6.1到6.3，回收率随pH的升高而升高，pH从6.3到6.7，回收率随pH的变化并无明显区别，提取溶液pH从6.7到6.9，回收率随pH的升高而降低。因此，确定提取液pH为6.5±0.2。ABCDEA.AMP；B.IMP；C.GMP；D.HxR；E.Hx图15提取液最终pH范围对加标回收率的影响(n=3)提取次数的确定从之前的优化结果看，最优回收率约为80%，回收率仍旧偏低，为了提高回收率，考虑通过增加提取次数的方式来提高回收率。实验结果如图16。提取一次的回收率为83%，提取两次的回收率为90%，提取三次的回收率为91%，提取两次和提取三次的回收率没有明显差异。因此，实验采用两次提取方式，实验最终回收率达到91%。图16提取次数对加标回收率的影响(n=3)净化方式的确定样品经过提取后，上清液中还含有大量油脂和蛋白质等杂质，杂质的分子质量较大，在流动相中流动缓慢，极易堵塞色谱柱，还会对目标物的检测产生基质干扰，降低方法的灵敏度。为了能有效对样品进行净化，除去油脂、蛋白质等基质干扰，需要选择合适的净化方式。实验中选择了固相萃取柱法、液液萃取法2种方式进行样品净化。经查阅文献，选择了MCX柱、HLB柱、WAX柱、PSA柱等不同固相萃取柱类型。实验发现，由于5种核苷酸的化学性质差异较大，没有一种固相萃取柱可以对5种核苷酸均有较好保留。使用通过型PRIMEHLB固相萃取柱，发现过柱后的提取液并未变得澄清，色谱图中杂质峰也未减少，而且会导致样品平行性变差。后采用正己烷液液萃取法除去杂质，经正己烷净化后的提取溶液变更澄清，实验结果见图17，同时核苷酸的回收率没有减小。因此，净化方式采用正己烷液液萃取法。未除脂的提取液除脂后的提取液未除脂的提取液除脂后的提取液图17经正己烷液液萃取法除杂质前后提取液状态的比较方法学考察标准曲线、检出限及定量限标准储备液（1mg/mL）：分别精密称定HxR、IMP、GMP、AMP、Hx约25mg（精确至0.00001g），用水溶解后，定容至25mL容量瓶中。配制成浓度均为1mg/mL的标准储备液，于4℃保存，有效期3个月。混合标准工作液（100μg/mL）：分别准确移取各标准储备液5mL，用水稀释并定容于50mL容量瓶中，配制成浓度均为100μg/mL的标准工作液，于4℃保存，有效期1个月。混合标准系列工作溶液：准确吸取混合标准工作液0.0500、0.500、2.00、5.00、8.00mL至10mL容量瓶，用水定容，配制成质量浓度为0.500、5.00、20.0、50.0、80.0μg/mL的混合标准系列工作溶液，临用现配。用优化好的液相色谱条件测定系列标准工作液。以测得峰面积（Y）为纵坐标，对应的标准溶液浓度（X）为横坐标，绘制标准曲线。其回归方程和决定系数如表1。5种核苷酸在0.5～80µg/mL浓度范围内呈现良好的线性关系。表15种核苷酸的回归方程和相关系数物质线性范围线性方程决定系数R2AMP0.5～80µg/mLY=0.1867X+0.00850.9998GMPY=0.1585X+0.00140.9997IMPY=0.1585X-0.00030.9999HxRY=0.2298X-0.00270.9999HxY=0.3580X+0.00740.9996本方法选取了猪肉脯、酱牛肉、腊肉、火腿肠、烧鸡作为代表样品，添加适量标准工作液进行试验，考察方法的检出限和定量限。经回收率试验，当添加量为5mg/kg时，信噪比（RS/N）均大于3；当添加量为15mg/kg时，信噪比（RS/N）均大于10，色谱图见图18。所以本方法的检出限确定为5.0mg/kg，定量限确定为15.0mg/kg。ABA.检出限；B.定量限图18检出限及定量限的色谱图稳定性考察按照设计好的时间，将配制的5种核苷酸的储备液于4℃冰箱保存，根据1个月、2个月、3个月、4个月、5个月、6个月的保存时间进行取样并分析，记录化合物峰面积。按以下公式计算剩余分析物（%）。剩余分析物（%）=Ci/C0×100%。其中：Ci=不同时间点的响应值；C0=新配标准储备溶液的第一次测得的响应值。根据表2的数据变化，储备液在三个月之内，响应值变化较小，结果表明，标准储备液有效期定为3个月是可行的。表2标准储备液剩余分析物的变化物质1个月2个月3个月4个月5个月6个月AMP99.599.298.396.591.286.7GMP99.799.198.595.691.688.5IMP99.399.299.096.390.283.6HxR99.298.998.695.692.185.6Hx99.998.598.896.191.181.9将储备液稀释成浓度为100μg/mL的混合标准工作液，并于4℃冰箱保存，按照0.5个月、1个月、1.5个月、2个月的保存时间进行取样并分析，记录化合物峰面积。按以下公式计算剩余分析物（%）。剩余分析物（%）=Ci/C0×100%。其中：Ci=不同时间点的响应值；C0=新配标准储备溶液的第一次测得的响应值。根据表3的数据变化，混合标准工作液在1个月之内，响应值变化较小。因此，混合标准工作液有效期定为1个月是可行的。表3标准工作液剩余分析物的变化物质0.5个月1个月1.5个月2个月AMP99.698.195.688.6GMP99.898.895.789.2IMP99.798.293.890.3HxR99.598.793.190.1Hx99.998.995.789.7方法的回收率按照GB/T19480-2025《肉与肉制品术语》中对肉制品的分类，选取不同加工工艺以及居民消费频率，本实验选取了猪肉脯、酱牛肉、腊肉、火腿肠、烧鸡作为代表样品，在样品中添加15mg/kg、30mg/kg、150mg/kg（LOQ、2LOQ、10LOQ）三个不同浓度的核苷酸进行回收率试验，每个浓度进行6个平行试验，重复进行3次，实验色谱图见图19-23，数据结果见表4-8。在本实验室内，15mg/kg～150mg/kg添加浓度范围的回收率为80.0%-109.5%。批内相对标准偏差≤9.8%，批间相对标准偏差≤7.7%。猪肉脯基质猪肉脯基质猪肉脯基质加标质加标图19猪肉脯基质及基质加标的色谱图酱牛肉基质酱牛肉基质酱牛肉基质加标图20酱牛肉基质及基质加标的色谱图腊肉基质加标腊肉基质加标腊肉基质加标图21腊肉基质及基质加标的色谱图火腿肠基质火腿肠基质火腿肠基质加标图22火腿肠基质及基质加标的色谱图烧鸡基质加标烧鸡基质加标烧鸡基质加标图23烧鸡基质及基质加标的色谱图

表4实验室内肌苷回收率测定结果待测组织添加浓度(mg/kg)批次回收率（%）批内平均回收率（%）批内RSD（%）批间平均回收率（%）批间RSD（%）123456猪肉脯15Ⅰ93.995.988.180.690.691.090.05.990.35.1Ⅱ85.381.392.789.481.685.185.95.2Ⅲ84.5104.0103.493.096.888.695.18.330Ⅰ93.694.9100.486.399.794.895.05.393.92.5Ⅱ88.685.193.8103.180.995.891.28.8Ⅲ104.6102.996.388.098.183.795.68.6150Ⅰ99.4100.794.992.896.796.796.93.093.13.7Ⅱ94.385.291.790.986.891.390.03.8Ⅲ95.193.994.183.099.588.692.46.2酱牛肉15Ⅰ84.196.490.099.182.890.490.57.293.63.2Ⅱ89.192.3103.896.5102.394.096.36.0Ⅲ96.684.597.599.495.291.194.15.830Ⅰ90.9101.295.4101.695.491.896.14.795.03.9Ⅱ94.7101.686.487.693.581.190.88.0Ⅲ100.997.394.791.7103.5100.398.14.4150Ⅰ89.499.587.497.497.199.195.05.593.53.9Ⅱ90.899.286.297.6101.9101.396.26.6Ⅲ97.882.888.092.890.484.789.46.1腊肉15Ⅰ98.888.584.7103.298.685.093.18.696.63.3Ⅱ104.697.9102.894.990.492.497.25.8Ⅲ97.2105.1100.7103.294.995.399.44.330Ⅰ95.1104.196.9100.0105.391.798.95.397.02.0Ⅱ96.4102.791.191.6102.199.597.25.2Ⅲ97.698.990.096.196.390.594.93.9150Ⅰ91.089.787.396.8100.294.793.35.294.53.1Ⅱ100.2104.6100.790.194.896.797.95.2Ⅲ84.592.1104.189.296.488.492.57.5火腿肠15Ⅰ93.691.290.089.8101.7103.995.06.593.21.9Ⅱ92.885.390.792.794.693.291.63.6Ⅲ95.187.493.088.696.996.392.94.330Ⅰ87.391.993.486.194.195.291.34.192.34.1Ⅱ94.886.487.184.993.487.589.04.6Ⅲ104.0104.496.092.091.091.496.56.5150Ⅰ99.282.6103.697.1106.598.898.08.596.81.5Ⅱ86.4101.791.887.9105.698.095.28.1Ⅲ90.088.0105.7104.491.3103.697.28.4烧鸡15Ⅰ103.7105.498.384.192.7102.597.88.395.85.1Ⅱ83.480.293.391.696.596.590.37.6Ⅲ97.2108.195.392.6108.795.099.57.130Ⅰ105.284.287.888.091.693.691.78.095.83.8Ⅱ104.2102.8103.497.1100.285.298.87.2Ⅲ103.489.6101.291.798.795.796.75.6150Ⅰ91.489.8104.395.6105.694.596.96.893.03.7Ⅱ101.592.394.990.686.785.691.96.3Ⅲ96.993.388.795.981.385.690.36.8表5实验室内肌苷酸回收率测定结果待测组织添加浓度(mg/kg)批次回收率（%）批内平均回收率（%）批内RSD（%）批间平均回收率（%）批间RSD（%）123456猪肉脯15Ⅰ97.186.785.184.892.188.489.05.495.15.6Ⅱ101.1100.396.6105.193.595.898.74.3Ⅲ101.597.895.294.8104.792.097.74.830Ⅰ101.391.589.298.583.7102.894.58.092.62.6Ⅱ98.096.195.698.080.291.893.37.3Ⅲ80.884.696.691.490.995.289.96.8150Ⅰ102.2100.294.681.6101.598.096.48.095.43.6Ⅱ90.583.888.097.392.997.491.75.8Ⅲ96.093.298.5100.299.5102.298.33.3酱牛肉15Ⅰ93.882.692.097.496.590.792.25.894.13.4Ⅱ84.695.9105.2103.696.4101.297.87.6Ⅲ98.694.389.291.085.496.092.45.230Ⅰ103.697.397.495.095.995.497.43.396.16.3Ⅱ83.980.896.686.999.089.689.58.0Ⅲ101.999.4102.1101.5105.397.7101.32.6150Ⅰ96.2101.196.796.395.798.597.42.195.53.1Ⅱ97.395.8103.795.398.790.997.04.4Ⅲ89.190.398.790.197.487.392.25.1腊肉15Ⅰ99.284.095.881.985.792.889.97.890.73.3Ⅱ88.796.991.195.798.193.394.03.8Ⅲ87.792.190.493.085.181.088.25.230Ⅰ102.292.597.2103.6102.584.797.17.694.04.1Ⅱ90.299.697.490.5102.890.395.15.8Ⅲ96.299.582.686.887.884.789.67.5150Ⅰ103.0103.197.495.298.593.098.44.294.64.2Ⅱ96.180.893.394.290.488.190.56.1Ⅲ86.083.0102.999.599.898.394.98.7火腿肠15Ⅰ99.386.296.590.8104.097.795.86.695.12.2Ⅱ91.994.8109.595.094.395.396.86.6Ⅲ94.390.991.094.8102.184.092.96.430Ⅰ89.396.490.487.391.293.991.43.691.50.9Ⅱ93.099.590.287.089.794.692.34.8Ⅲ98.089.291.088.487.690.590.84.1150Ⅰ98.990.390.087.390.1104.993.67.394.03.8Ⅱ105.898.2100.390.986.9104.997.87.7Ⅲ91.887.697.386.189.891.390.74.3烧鸡15Ⅰ97.9100.1100.696.888.386.895.16.392.02.9Ⅱ99.283.6100.783.382.196.991.09.7Ⅲ92.383.787.097.094.585.790.05.930Ⅰ103.592.799.4103.288.683.095.18.892.85.6Ⅱ104.890.993.298.386.7105.096.57.8Ⅲ84.583.192.980.494.885.686.96.6150Ⅰ97.588.689.6102.683.488.291.77.792.52.3Ⅱ94.097.6100.790.891.794.494.93.9Ⅲ87.487.985.1103.191.390.290.87.0表6实验室内鸟苷酸回收率测定结果待测组织添加浓度(mg/kg)批次回收率（%）批内平均回收率（%）批内RSD（%）批间平均回收率（%）批间RSD（%）123456猪肉脯15Ⅰ97.485.081.186.190.888.088.16.491.43.6Ⅱ98.686.098.886.691.588.291.66.3Ⅲ91.290.193.296.3102.794.194.64.830Ⅰ87.792.599.490.391.597.693.24.894.31.4Ⅱ80.996.998.293.096.199.594.17.3Ⅲ98.797.795.5102.882.397.495.77.3150Ⅰ97.992.793.299.397.792.995.63.190.74.7Ⅱ93.490.881.482.481.697.387.87.9Ⅲ88.997.483.384.785.792.788.86.1酱牛肉15Ⅰ83.687.690.395.891.490.489.94.591.34.1Ⅱ80.283.399.588.486.193.888.68.0Ⅲ90.296.085.5103.2103.195.195.57.330Ⅰ85.990.795.890.387.698.691.55.393.22.8Ⅱ101.7101.493.196.289.395.696.25.0Ⅲ97.393.097.381.796.985.692.07.3150Ⅰ90.294.196.793.694.886.592.74.093.83.3Ⅱ92.483.489.3103.284.196.091.48.2Ⅲ103.485.8100.2103.598.193.197.47.0腊肉15Ⅰ82.991.790.294.091.189.990.04.294.34.3Ⅱ92.9108.099.099.792.994.997.95.9Ⅲ103.590.394.791.690.2100.095.15.830Ⅰ91.295.595.896.997.595.295.42.391.24.3Ⅱ80.484.881.593.297.388.987.77.6Ⅲ94.388.083.4101.986.089.090.47.4150Ⅰ81.096.785.897.786.188.289.37.492.74.0Ⅱ88.195.996.093.9104.3101.096.55.8Ⅲ92.491.790.7102.987.288.592.26.1火腿肠15Ⅰ103.495.099.590.298.097.397.24.694.22.8Ⅱ88.696.6103.188.387.592.092.76.6Ⅲ87.386.499.097.594.191.392.65.630Ⅰ97.195.591.093.2104.894.396.05.093.72.2Ⅱ103.396.294.380.386.598.293.19.0Ⅲ80.584.798.689.998.599.792.08.9150Ⅰ102.492.492.7102.386.883.393.38.491.02.6Ⅱ84.1102.480.090.098.292.091.19.2Ⅲ100.496.584.583.685.580.888.68.9烧鸡15Ⅰ84.488.584.590.294.298.490.06.192.12.1Ⅱ103.790.090.887.795.787.992.66.6Ⅲ95.993.487.297.198.790.093.74.730Ⅰ85.185.7101.188.096.091.591.26.993.62.5Ⅱ85.592.197.295.996.394.493.64.6Ⅲ86.798.597.490.998.2104.396.06.5150Ⅰ86.882.697.186.793.985.788.86.289.93.6Ⅱ84.392.597.087.782.280.687.47.3Ⅲ95.290.291.793.993.397.193.62.6表7实验室内腺苷酸回收率测定结果待测组织添加浓度(mg/kg)批次回收率（%）批内平均回收率（%）批内RSD（%）批间平均回收率（%）批间RSD（%）123456猪肉脯15Ⅰ97.186.785.184.882.188.487.46.089.33.4Ⅱ81.1100.396.685.197.595.892.78.3Ⅲ81.587.895.284.884.792.087.75.830Ⅰ81.391.589.288.583.782.886.24.891.25.0Ⅱ98.096.195.698.090.291.895.03.4Ⅲ100.889.696.681.490.995.292.47.3150Ⅰ82.290.294.681.691.598.089.77.491.05.6Ⅱ80.583.888.087.392.987.486.74.8Ⅲ96.093.298.590.299.5102.296.64.5酱牛肉15Ⅰ83.892.692.087.486.580.787.25.392.55.8Ⅱ94.695.995.2103.696.4101.297.83.8Ⅲ98.694.389.291.085.496.092.45.230Ⅰ103.687.387.495.095.995.494.16.594.16.7Ⅱ83.980.896.686.989.089.687.86.2Ⅲ101.999.4102.1101.5100.397.7100.51.7150Ⅰ96.2101.196.796.395.786.595.45.094.32.0Ⅱ97.395.8103.795.388.790.995.35.5Ⅲ89.190.398.790.197.487.392.25.1腊肉15Ⅰ99.2104.095.891.995.792.896.64.691.94.9Ⅱ88.786.981.195.780.193.387.67.2Ⅲ87.792.190.493.085.1101.091.66.030Ⅰ102.292.597.298.6102.594.798.04.193.75.5Ⅱ90.299.697.490.5102.890.395.15.8Ⅲ96.289.582.686.887.884.787.95.4150Ⅰ83.0103.197.485.290.593.092.08.294.13.8Ⅱ96.180.893.394.290.498.192.26.7Ⅲ86.0103.0102.999.599.898.398.36.4火腿肠15Ⅰ89.386.296.590.884.087.789.14.992.43.1Ⅱ91.994.889.595.094.395.393.52.5Ⅲ94.390.991.094.8102.194.094.54.330Ⅰ89.386.490.487.391.283.988.13.191.53.4Ⅱ83.099.5100.287.089.794.692.37.5Ⅲ98.089.291.088.497.6100.594.15.5150Ⅰ88.990.390.087.390.1104.991.97.092.42.1Ⅱ95.898.290.390.986.9104.994.56.9Ⅲ81.897.697.386.189.891.390.76.8烧鸡15Ⅰ97.9100.180.686.888.386.890.18.290.92.4Ⅱ99.283.690.783.382.196.989.38.4Ⅲ92.383.787.097.094.5105.793.48.330Ⅰ83.592.799.4103.288.683.091.79.191.73.4Ⅱ94.890.993.298.386.7105.094.86.7Ⅲ94.583.192.980.494.885.688.67.1150Ⅰ97.588.689.6102.683.488.291.77.792.45.0Ⅱ84.097.6100.780.881.784.488.29.8Ⅲ97.497.995.1103.191.399.297.34.1表8实验室内次黄嘌呤回收率测定结果待测组织添加浓度(mg/kg)批次回收率（%）批内平均回收率（%）批内RSD（%）批间平均回收率（%）批间RSD（%）123456猪肉脯15Ⅰ97.196.785.184.8102.198.494.07.891.67.7Ⅱ81.180.386.685.183.585.883.73.1Ⅲ91.597.895.294.8104.799.097.24.730Ⅰ91.381.589.288.583.792.887.85.091.53.9Ⅱ98.096.195.698.090.291.895.03.4Ⅲ100.884.696.681.490.995.291.68.1150Ⅰ92.290.294.681.691.598.091.46.093.63.9Ⅱ90.583.888.097.392.997.491.75.8Ⅲ96.093.298.597.299.5102.297.83.2酱牛肉15Ⅰ83.882.692.087.486.590.787.24.291.94.9Ⅱ94.695.995.293.696.4101.296.22.8Ⅲ98.694.389.291.085.496.092.45.230Ⅰ93.697.397.495.095.995.495.81.591.65.4Ⅱ83.980.886.686.989.089.686.13.8Ⅲ101.999.492.181.585.397.793.08.8150Ⅰ96.2101.196.796.395.798.597.42.193.34.6Ⅱ97.395.883.795.398.790.993.65.9Ⅲ89.190.388.790.187.487.388.81.4腊肉15Ⅰ99.294.095.881.985.792.891.67.190.85.0Ⅱ88.786.991.185.780.183.386.04.5Ⅲ87.792.1100.493.095.1101.094.95.430Ⅰ102.292.597.293.692.594.795.53.990.65.1Ⅱ80.299.697.490.592.880.390.19.2Ⅲ86.289.582.686.887.884.786.32.8150Ⅰ103.093.197.495.290.593.095.44.694.74.1Ⅱ86.180.893.394.290.498.190.56.9Ⅲ96.093.0102.999.599.898.398.33.5火腿肠15Ⅰ99.386.296.590.884.087.790.86.691.22.2Ⅱ91.994.889.595.094.395.393.52.5Ⅲ94.390.981.094.892.184.089.56.430Ⅰ89.396.490.487.391.293.991.43.689.82.3Ⅱ83.099.590.287.089.794.690.76.4Ⅲ88.089.291.088.487.680.587.54.1150Ⅰ98.990.390.087.390.184.990.35.291.21.5Ⅱ85.898.2100.390.986.994.992.86.4Ⅲ91.887.697.386.189.891.390.74.3烧鸡15Ⅰ97.9100.1100.696.888.386.895.16.392.02.9Ⅱ99.283.6100.783.382.196.991.09.7Ⅲ92.383.787.097.094.585.790.05.930Ⅰ103.592.799.493.288.683.093.47.990.03.2Ⅱ84.880.993.298.386.785.088.27.3Ⅲ94.583.192.980.494.885.688.67.1150Ⅰ97.588.689.692.683.488.290.05.390.84.1Ⅱ94.097.6100.790.891.794.494.93.9Ⅲ87.487.985.183.191.390.287.53.5实验室内的结果验证试验实验室内部组织双人双仪器进行方法学验证，以猪肉脯、酱牛肉、腊肉、火腿肠、烧鸡作为代表样品，在样品中添加15mg/kg、30mg/kg、150mg/kg（LOQ、2LOQ、10LOQ）三个不同浓度的核苷酸进行回收率试验，每个浓度进行6个平行试验，数据结果见表9。实验室内部组织双人双仪器，15mg/kg～150mg/kg添加浓度范围的平均回收率为85.4%-101.8%。批内相对标准偏差≤9.6%，批间相对标准偏差≤8.1%。表9实验室双人双仪器验证实验结果待测组织添加浓度(mg/kg)人员平均回收率（%）RSD(%)平均回收率(%)双人双仪器RSD(%)猪肉脯15人员194.07.891.67.7人员283.73.130人员187.85.091.53.9人员295.03.493.63.9150人员191.68.1人员291.46.0酱牛肉15人员191.75.891.94.9人员297.83.230人员187.24.2人员296.22.891.65.4150人员192.45.2人员295.81.5腊肉15人员186.13.893.34.6人员293.08.830人员197.42.1人员293.65.990.85.0150人员188.81.4人员291.67.1火腿肠15人员186.04.590.65.1人员294.95.430人员195.53.9人员290.19.294.74.1150人员186.32.8人员295.44.6烧鸡15人员190.56.991.22.2人员298.33.530人员190.86.6人员293.52.589.82.3150人员189.56.4人员291.43.6

实验室间的结果验证试验方法标准制定完成后，标准编制小组委托山东省食品药品检验研究院、浙江工商大学海洋食品研究院、中国海关科学技术研究中心三家单位对方法标准的灵敏度、准确度、精密度和不同基质的适用性进行了验证，验证结论为该方法可操作性强，准确度高、重复性好，满足药残检测的要求。各单位的验证结果及实验室间的重复性结果见表10～表24，验证报告扫描件见附录A。

表10山东院肌苷回收率测定结果待测组织添加浓度(mg/kg)批次回收率（%）批内平均回收率（%）批内RSD（%）批间平均回收率（%）批间RSD（%）123456猪肉脯15Ⅰ101.3100.096.094.787.381.393.48.394.32.0Ⅱ90.7100.792.784.792.097.393.06.0Ⅲ103.3101.3102.788.786.796.096.47.630Ⅰ94.081.786.089.793.098.090.46.592.21.7Ⅱ93.387.795.089.791.3102.793.35.7Ⅲ103.089.386.098.395.385.792.97.6150Ⅰ93.389.185.383.581.889.687.14.992.68.7Ⅱ102.1106.6104.392.2105.1100.6101.85.1Ⅲ82.990.092.794.890.082.288.85.8酱牛肉15Ⅰ92.798.096.084.086.094.091.86.190.12.5Ⅱ84.084.784.081.398.093.387.67.5Ⅲ85.393.391.3100.786.788.791.06.130Ⅰ102.795.390.094.392.098.095.44.792.13.2Ⅱ86.793.390.784.0100.785.390.16.9Ⅲ93.096.395.084.791.084.090.75.8150Ⅰ91.5101.5105.889.8102.197.598.06.494.63.6Ⅱ104.991.790.792.592.395.194.55.6Ⅲ89.281.492.5102.390.292.091.37.4腊肉15Ⅰ94.792.081.384.790.786.788.35.689.22.8Ⅱ90.090.083.392.785.382.087.24.9Ⅲ90.089.3102.094.788.088.092.06.030Ⅰ98.384.393.091.094.084.790.96.190.12.1Ⅱ94.783.798.0100.388.783.391.48.0Ⅲ94.091.380.787.788.385.387.95.3150Ⅰ96.289.495.282.699.592.392.56.490.32.2Ⅱ87.988.287.585.9100.782.088.77.1Ⅲ93.389.398.586.385.585.489.75.9火腿肠15Ⅰ94.086.787.394.095.386.790.74.692.62.1Ⅱ95.385.389.399.399.398.794.66.3Ⅲ93.3103.384.092.791.390.792.66.730Ⅰ101.397.390.087.391.395.093.75.590.45.2Ⅱ90.095.398.788.397.785.392.65.9Ⅲ80.384.786.086.086.786.385.02.8150Ⅰ95.196.384.6102.197.0105.196.77.395.12.6Ⅱ93.986.788.185.1102.397.392.27.3Ⅲ93.291.395.6106.193.998.496.45.5烧鸡15Ⅰ91.382.796.083.384.098.789.37.890.14.1Ⅱ84.083.396.782.088.087.386.96.1Ⅲ96.086.7103.386.799.393.394.27.230Ⅰ96.791.7103.089.796.386.794.06.292.91.1Ⅱ97.382.0100.789.398.088.392.67.8Ⅲ91.795.388.096.780.7100.392.17.6150Ⅰ106.2102.7103.593.698.3103.2101.24.596.24.6Ⅱ91.793.294.988.999.789.793.04.2Ⅲ90.096.792.498.295.193.194.33.2表11山东院肌苷酸回收率测定结果待测组织添加浓度(mg/kg)批次回收率（%）批内平均回收率（%）批内RSD（%）批间平均回收率（%）批间RSD（%）123456猪肉脯15Ⅰ93.386.788.092.094.791.391.03.493.63.1Ⅱ88.797.3102.7100.0101.390.796.86.0Ⅲ92.794.788.794.793.394.093.02.430Ⅰ95.391.389.080.380.384.086.77.191.44.8Ⅱ85.097.795.094.797.3102.795.46.1Ⅲ89.0102.395.792.782.390.792.17.3150Ⅰ95.499.9102.1105.3102.7105.5101.83.7100.81.9Ⅱ98.398.7102.996.4100.394.998.62.9Ⅲ103.197.2106.097.0106.7101.5101.94.1酱牛肉15Ⅰ90.098.0103.398.7102.094.797.85.096.51.2Ⅱ93.395.399.392.096.796.095.42.7Ⅲ101.3102.797.388.794.792.796.25.530Ⅰ102.780.791.096.7102.084.392.99.992.90.1Ⅱ83.387.085.7103.397.0101.392.99.3Ⅲ102.392.783.794.395.089.092.86.7150Ⅰ96.195.9105.098.7101.594.498.64.198.40.4Ⅱ101.895.3102.499.198.894.998.73.2Ⅲ99.395.998.1102.795.096.798.02.9腊肉15Ⅰ86.790.088.098.086.7100.791.76.795.33.8Ⅱ103.399.3101.3103.386.0100.098.96.6Ⅲ101.390.797.393.398.092.095.44.330Ⅰ100.795.792.093.098.083.093.76.692.92.6Ⅱ87.780.783.088.3102.399.390.29.7Ⅲ99.089.091.097.789.7102.394.85.9150Ⅰ98.7100.094.598.395.193.996.82.698.83.3Ⅱ94.795.598.194.4103.396.597.13.4Ⅲ105.0103.795.6106.4102.1102.9102.63.7火腿肠15Ⅰ100.096.799.394.094.097.396.92.694.22.5Ⅱ101.398.790.392.087.387.392.86.4Ⅲ96.090.788.096.097.389.392.94.330Ⅰ99.394.093.099.390.381.392.97.292.02.8Ⅱ94.085.398.780.386.390.089.17.4Ⅲ89.795.087.390.7100.3101.394.16.2150Ⅰ95.996.3105.1103.0106.3106.6102.24.8101.30.8Ⅱ99.996.1105.9105.995.1101.6100.74.6Ⅲ100.795.3104.1106.5103.195.4100.84.6烧鸡15Ⅰ87.386.786.090.790.092.088.82.791.53.0Ⅱ88.786.789.389.3100.794.091.45.6Ⅲ94.798.794.093.398.087.394.34.330Ⅰ100.087.087.084.380.092.088.47.891.32.8Ⅱ99.387.380.086.3101.0100.792.49.8Ⅲ84.0103.097.795.780.798.093.29.4150Ⅰ95.996.994.797.794.994.695.81.499.63.4Ⅱ94.2102.495.4104.8106.3100.5100.64.9Ⅲ105.598.1106.1105.7100.598.7102.43.7表12山东院鸟苷酸回收率测定结果待测组织添加浓度(mg/kg)批次回收率（%）批内平均回收率（%）批内RSD（%）批间平均回收率（%）批间RSD（%）123456猪肉脯15Ⅰ103.396.0102.794.097.396.098.23.994.63.6Ⅱ88.799.3100.794.787.393.394.05.8Ⅲ91.393.394.094.788.087.391.43.430Ⅰ103.397.0101.7102.3100.798.7100.62.499.01.7Ⅱ102.798.7101.094.3101.396.799.13.2Ⅲ99.3103.395.395.796.093.797.23.6150Ⅰ104.9101.5105.397.198.395.9100.54.099.71.4Ⅱ94.3101.0100.799.495.398.198.12.9Ⅲ104.998.299.896.4104.998.7100.53.6酱牛肉15Ⅰ88.090.0102.0100.795.388.794.16.696.23.2Ⅱ103.3100.7101.3100.7102.090.799.84.6Ⅲ98.793.387.3100.786.7101.394.76.930Ⅰ100.093.7101.398.096.394.097.23.298.21.0Ⅱ101.798.397.794.395.0102.098.23.3Ⅲ97.396.398.096.7103.3103.099.13.2150Ⅰ103.595.793.7100.2102.897.798.93.999.20.7Ⅱ98.693.7103.395.6102.998.498.73.9Ⅲ97.9104.6101.5100.393.4102.1100.03.9腊肉15Ⅰ99.386.7101.399.391.393.395.26.096.20.9Ⅱ89.391.3100.796.7102.798.796.65.4Ⅲ101.396.096.0102.786.798.796.95.930Ⅰ98.396.0102.0100.3102.7101.7100.22.599.60.5Ⅱ96.799.0100.0102.099.398.799.31.8Ⅲ98.3100.7100.794.399.0102.799.32.9150Ⅰ97.9100.795.3102.494.499.998.43.299.71.9Ⅱ98.5105.599.7105.798.3104.0101.93.4Ⅲ95.794.9105.198.3102.097.098.84.0火腿肠15Ⅰ100.092.796.7102.093.387.395.35.693.81.4Ⅱ100.788.090.386.796.797.393.36.1Ⅲ96.093.395.390.789.392.792.92.830Ⅰ100.795.3103.394.0100.7100.799.13.797.81.7Ⅱ97.099.095.3102.395.0101.798.43.2Ⅲ101.796.793.396.393.394.395.93.3150Ⅰ94.398.1105.3103.5100.0102.7100.74.0101.91.6Ⅱ106.1105.1104.995.9106.3104.1103.73.8Ⅲ97.7102.798.7104.599.3105.7101.43.3烧鸡15Ⅰ102.789.390.7103.3103.3100.798.36.796.02.6Ⅱ102.0101.398.788.0101.386.796.37.3Ⅲ99.392.094.788.790.794.793.34.030Ⅰ93.394.396.0100.795.0103.397.14.197.70.6Ⅱ96.097.3100.399.097.097.797.91.6Ⅲ96.793.799.099.3100.0100.798.22.7150Ⅰ95.095.697.1104.1102.298.098.73.799.61.1Ⅱ104.599.194.5103.197.7105.9100.84.4Ⅲ95.2105.1101.7104.194.795.099.34.9表13山东院腺苷酸回收率测定结果待测组织添加浓度(mg/kg)批次回收率（%）批内平均回收率（%）批内RSD（%）批间平均回收率（%）批间RSD（%）123456猪肉脯15Ⅰ101.396.098.7101.396.795.398.22.794.63.3Ⅱ94.093.390.097.389.390.092.33.4Ⅲ96.7100.086.794.787.394.793.35.730Ⅰ101.395.397.3100.095.394.097.23.097.82.0Ⅱ95.096.793.398.093.3100.796.23.0Ⅲ97.3103.0102.0102.399.096.3100.02.8150Ⅰ100.095.1105.995.3102.6105.0100.64.799.80.9Ⅱ98.395.095.3104.9101.398.598.93.8Ⅲ96.596.3101.298.1105.3102.199.93.6酱牛肉15Ⅰ100.797.3103.393.397.398.798.43.495.32.9Ⅱ92.794.096.794.794.091.393.91.9Ⅲ91.387.392.796.7100.792.093.45.030Ⅰ95.3103.396.3101.398.096.398.43.297.21.3Ⅱ95.393.7101.393.398.793.395.93.5Ⅲ98.393.798.0100.794.099.397.32.9150Ⅰ94.0101.297.4104.494.196.397.94.297.71.4Ⅱ103.395.394.395.2101.1104.899.04.7Ⅲ94.193.8100.096.596.296.796.22.3腊肉15Ⅰ90.797.3100.791.387.398.794.35.693.11.3Ⅱ88.094.793.394.093.394.092.92.6Ⅲ95.389.386.794.792.793.392.03.630Ⅰ101.3101.3103.398.094.098.099.33.498.80.5Ⅱ98.797.397.796.799.3100.098.31.3Ⅲ102.399.794.397.796.3102.098.73.2150Ⅰ94.9103.2105.496.197.7102.199.94.399.50.8Ⅱ98.3103.997.094.7100.797.298.63.3Ⅲ97.099.198.797.4105.3102.9100.13.3火腿肠15Ⅰ