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Mass Spectrometry Matrix Effect Study for the Trace Analysis of Chloramphenicol Residues in Honey
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摘要: 有机同位素稀释质谱法（Isotope dilution mass spectrometry，IDMS）是复杂基质样品中痕量成分准确测量的潜在基准方法，但分析条件、同位素稀释试剂、基质效应（Matrix Effect，ME)）等因素也会对结果产生影响。本文以枣花和荆花蜂蜜为研究样品，比较不同提取方法下蜂蜜中氯霉素痕量残留测量时的基质效应，研究基质效应对结果的影响、基质效应的补偿及消除方法。采用基质效应柱后注射定性评价方法和萃取后添加定量评价方法，考察直接稀释提取法和乙酸乙酯提取法的基质效应及其对氯霉素测量结果准确性的影响；结果表明，直接稀释提取溶液中氯霉素受到的基质抑制效应更为显著，ME在7%～10%之间，抑制了90%以上的氯霉素质谱响应，外标法校准的提取回收率仅为10.9%±1.9%；乙酸乙酯法提取溶液的ME在33%～35%之间，基体抑制相对较弱，外标法校准的提取回收率为33.7%±1.2%。采用同位素稀释质谱法进行校准，两种提取方法的回收率分别为100.4%±4.3%、100.2%±3.4%，说明基质抑制效应对测量结果的影响被有效补偿。直接稀释提取法采用IDMS校准，可准确、快速测量蜂蜜中氯霉素痕量残留。
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关键词:
- 蜂蜜 /
- 氯霉素 /
- 基质效应 /
- 同位素稀释质谱法 /
- 液相色谱串联三重四级杆质谱（LC-MS/MS）
Abstract: Organic isotope dilution mass spectrometry (IDMS) is a potential reference method for the accurate measurement of trace components in complex matrix samples. However, the analytical conditions, isotope dilution reagents, matrix effects (MEs), and other factors can also affect the results. In this paper, jujube and wattle honey were used as the study samples to compare the MEs in the trace measurement of chloramphenicol residues in honey under different extraction methods, so as to investigate the influence of MEs on the results, the compensation and elimination methods of MEs. -
图 2 不同方法提取液影响CAP质谱响应的总离子流
注：a1.空白溶剂(15%甲醇水溶液）；b1.乙酸乙酯法提取的空白蜂蜜提取溶液；c1.直接稀释法提取的空白蜂蜜溶液；d1. CAP 的LC-MS/MS谱图。
Figure 2. The total ion chromatogram of different solutions affecting the response of CAP mass spectrometry
图 3 不同体积水淋洗液影响CAP质谱响应的总离子流
注：a.空白溶剂（15%甲醇水溶液）；b.20 ml水淋洗；c.10 ml水淋洗；d. 5 ml 水淋洗；e.CAP甲醇溶液 LC-MS/MS质谱图
Figure 3. The total ion chromatogram of the effect of different volume of water eluent on the response of CAP
图 4 不同比例甲醇水淋洗液影响CAP质谱响应的离子流
注：a.空白溶剂（15%甲醇水）； b.50%甲醇水淋洗；c.25%甲醇水淋洗；d.5%甲醇水淋洗；e.CAP 甲醇溶液LC-MS/MS质谱图
Figure 4. The total ion chromatogram of the effect of different proportions of methanol-water on the response of CAP mass spectrometry
图 5 两种前处理方法提取步骤及耗时情况 (n=100) (A) 直接稀释法（B）乙酸乙酯提取法
Figure 5. Extraction steps and time consuming of two pre-processing methods (n = 100) (A) Direct dilution method (B) Ethyl acetate extraction method
表 1 LC-MS/MS检测离子对信息
Table 1. Transition reactions monitored by LC- MS/MS
分析物 保留时间/min 母离子(m/z) 子离子(m/z) 碰撞电压/V CAP 3.4 321 152* −17 194 −11 257 −10 D5-CAP 3.4 326 157* −17 199 −11 262 −10 *定量离子 
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表 2 直接稀释法与乙酸乙酯提取法应用于不同蜂蜜基质中的ME与θ值（n=3）
Table 2. ME and correction factor of direct dilution method and ethyl acetate extraction method applied to different honey（n =3）
基质效应 枣花蜂蜜 荆花蜂蜜 直接稀释法 MEcap/% 10.4 ± 0.7 7.6 ± 1.0 MEd5-cap/% 10.2 ± 0.9 7.6 ± 1.1 θ 1.03 ± 0.03 1.01 ± 0.04 乙酸乙酯提取法 MEcap/% 35.2 ± 2.6 33.3 ± 4.0 MEd5-cap/% 36.3 ± 2.5 34.5 ± 3.8 θ 0.97 ± 0.02 0.97 ± 0.03
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表 3 两种提取溶液不同净化条件的基质效应（n=6）
Table 3. Matrix effect optimized by two extraction methods (n = 6)
优化条件 直接稀释法 乙酸乙酯提取法 MEcap/% MEd5-cap/% MEcap/% MEd5-cap/% 水体积/mL 20 11.2 ± 0.6 11.6 ± 0.8 11.8 ± 1.0 11.5 ± 0.7 10 12.4 ± 0.9 12.8 ± 1.1 12.2 ± 1.5 12.3 ± 1.5 5 12.0 ± 1.0 12.0 ± 1.1 12.3 ± 1.0 12.2 ± 0.7 甲醇比例/% 50 11.9 ± 0.7 12.3 ± 0.7 21.5 ± 2.2 21.1 ± 1.9 25 6.5 ± 0.8 6.6 ± 0.8 13.2 ± 1.7 12.5 ± 1.9 5 4.1 ± 0.7 4.5 ± 1.1 12.5 ± 0.9 12.1 ± 1.0
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表 4 直接稀释法和乙酸乙酯提取法提取枣花蜂蜜中的CAP回收率%（n=6）
Table 4. Recovery% of CAP in jujube honey by direct dilution method and ethyl acetate extraction method (n = 6)
浓度/μg·kg1 直接稀释法 乙酸乙酯提取法 RE1 RE2 RE3 RE4 RE1 RE2 RE3 RE4 0.1 10.7 ± 2.4 99.1 ± 4.8 101.8 ± 4.6 100.1 ± 4.9 31.3 ± 1.0 69.8 ± 5.2 101.1 ± 3.1 103.8 ± 4.7 0.4 11.3 ± 1.4 103.1 ± 3.2 102.2 ± 5.1 100.7 ± 4.4 30.0 ± 1.8 75.8 ± 4.3 97.8 ± 3.3 99.9 ± 2.8 2.0 10.8 ± 1.8 100.1 ± 2.5 100.6 ± 2.2 100.5 ± 3.8 33.8 ± 0.8 87.0 ± 3.0 97.6 ± 4.4 96.8 ± 2.9 平均回收率% 10.9 ± 1.9 100.8 ± 3.5 101.3 ± 3.9 100.4 ± 4.3 33.7 ± 1.2 77.6 ± 4.2 98.8 ± 3.6 100.2 ± 3.4 注：1.溶液外标校准法；2.基质匹配外标校准法；3. IDMS溶液校准法；4. IDMS基质匹配校准法
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表 5 两种提取方法应用于不同实际蜂蜜样本中CAP的检测浓度μg·kg−1（n=5）
Table 5. Detection concentration of CAP in different real honey samples by two extraction methods μg·kg−1 (n = 5)
蜂蜜 直接稀释法 乙酸乙酯提取法 荆花 0.0861±0.0032 0.0841±0.0049 枣花 0.0233±0.0019 0.0277±0.0024 枇杷 0.0619±0.0015 0.0561±0.0021 椴树 1.05±0.07 1.07±0.02
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