ICS67.050
CCSX 04
中華人民共和國(guó)出入境檢驗(yàn)檢疫行業(yè)標(biāo)準(zhǔn)
SN/T5639—2024
出口畜禽肉中松香酸和脫氫松香酸的測(cè)定
Determination of abietic acid and dehydroabietic acid in livestock and poultry
muscles for export
2024-12-16發(fā)布2025-06-01實(shí)施
中華人民共和國(guó)海關(guān)總署發(fā)布
前 言
本文件按照GB/T 1.1—2020《標(biāo)準(zhǔn)化工作導(dǎo)則 第1部分:標(biāo)準(zhǔn)化文件的結(jié)構(gòu)和起草規(guī)則》的規(guī)
定起草。
請(qǐng)注意本文件的某些內(nèi)容可能涉及專利。本文件的發(fā)布機(jī)構(gòu)不承擔(dān)識(shí)別專利的責(zé)任。
本文件由中華人民共和國(guó)海關(guān)總署提出并歸口。
本文件起草單位:中華人民共和國(guó)廣州海關(guān)、中國(guó)肉類(lèi)食品綜合研究中心、中國(guó)海關(guān)科學(xué)技術(shù)研究
中心。
本文件主要起草人:李菊、謝建軍、曾廣豐、古瑾、王璐、韓深、席靜、董潔、陳文銳、王志元、侯穎燁。
Ⅰ
SN/T5639—2024
出口畜禽肉中松香酸和脫氫松香酸的測(cè)定
1 范圍
本文件規(guī)定了出口畜禽肉中松香酸和脫氫松香酸含量的高效液相色譜測(cè)定方法及液相色譜-質(zhì)譜/
質(zhì)譜確證方法。
本文件適用于雞肉、鴨肉、鵝肉、豬肉、牛肉、羊肉中松香酸及脫氫松香酸殘留量的測(cè)定和確證,其他
畜禽肉類(lèi)食品可參照?qǐng)?zhí)行。
2 規(guī)范性引用文件
下列文件中的內(nèi)容通過(guò)文中的規(guī)范性引用而構(gòu)成本文件必不可少的條款。其中,注日期的引用文
件,僅該日期對(duì)應(yīng)的版本適用于本文件;不注日期的引用文件,其最新版本(包括所有的修改單)適用于
本文件。
GB/T 6682 分析實(shí)驗(yàn)室用水規(guī)格和試驗(yàn)方法
3 術(shù)語(yǔ)和定義
本文件沒(méi)有需要界定的術(shù)語(yǔ)和定義。
4 原理
試樣中的松香酸和脫氫松香酸用乙腈提取,經(jīng)硅膠鍵合C18 凈化劑凈化,用液相色譜二極管陣列或
紫外檢測(cè)器測(cè)定,外標(biāo)法定量,液相色譜-質(zhì)譜/質(zhì)譜法確證。
5 試劑和材料
除非另有規(guī)定,僅使用分析純?cè)噭?試驗(yàn)用水應(yīng)符合GB/T 6682中規(guī)定的一級(jí)水。
5.1 乙腈:色譜純。
5.2 甲醇:色譜純。
5.3 甲酸:色譜純
5.4 磷酸:優(yōu)級(jí)純。
5.5 無(wú)水硫酸鎂。
5.6 氯化鈉。
5.7 檸檬酸鈉。
5.8 檸檬酸氫二鈉。
5.9 0.1%磷酸水溶液:吸取1 mL磷酸(5.4),加水定容至1 000 mL,混勻。
5.10 松香酸 :純度大于等于98%,松香酸標(biāo)準(zhǔn)物質(zhì)的信息見(jiàn)附錄A 中表A.1。
5.11 脫氫松香酸 :純度大于等于98%,脫氫松香酸標(biāo)準(zhǔn)物質(zhì)的信息見(jiàn)附錄A 中表A.1。
5.12 松香酸標(biāo)準(zhǔn)儲(chǔ)備溶液(1 000 mg/L):準(zhǔn)確稱取10 mg松香酸標(biāo)準(zhǔn)品(5.10)于小燒杯中,加少量
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甲醇(5.2)溶解,移入10 mL容量瓶中,用甲醇定容至刻度,搖勻,-18 ℃避光保存,有效期為6個(gè)月。
5.13 脫氫松香酸標(biāo)準(zhǔn)儲(chǔ)備溶液(1 000 mg/L):精密稱取10 mg脫氫松香酸標(biāo)準(zhǔn)品(5.11)于小燒杯
中,加少量甲醇(5.2)溶解,移入10 mL容量瓶中,用甲醇定容至刻度,搖勻,-18 ℃避光保存,有效期為
6個(gè)月。
5.14 標(biāo)準(zhǔn)中間溶液(100.0 mg/L):分別量取1 mL標(biāo)準(zhǔn)儲(chǔ)備溶液(5.12和5.13),用乙腈(5.1)稀釋并
定容至10 mL,搖勻,-18 ℃避光保存,有效期為1個(gè)月。
5.15 標(biāo)準(zhǔn)工作溶液:準(zhǔn)確量取標(biāo)準(zhǔn)中間溶液(5.14),用乙腈提取液稀釋成0.10 mg/L、0.20 mg/L、0.
50 mg/L、1.00 mg/L、2.00 mg/L和5.00 mg/L的系列濃度標(biāo)準(zhǔn)工作溶液,現(xiàn)配現(xiàn)用。
5.16 硅膠鍵合C18 凈化劑:粒徑50 μm。
5.17 0.22 μm 微孔濾膜,有機(jī)系。
6 儀器和設(shè)備
6.1 高效液相色譜儀:配二極管陣列檢測(cè)器(PDA)或紫外檢測(cè)器。
6.2 高效液相色譜-質(zhì)譜/質(zhì)譜儀:配有電噴霧離子源(ESI)。
6.3 電子天平:感量為0.01 g和0.1 mg。
6.4 組織搗碎機(jī)。
6.5 渦旋混合器。
6.6 高速離心機(jī):轉(zhuǎn)速不低于4 000 r/min。
6.7 微型高速離心機(jī):轉(zhuǎn)速不低于10 000 r/min。
6.8 氮吹儀。
7 試樣的制備與保存
7.1 在制樣的操作過(guò)程中,應(yīng)防止樣品污染或發(fā)生殘留量含量的變化。
7.2 從所取樣品中取出有代表性樣品(有皮畜禽肉類(lèi)樣品帶皮)約500 g,用組織搗碎機(jī)搗碎,裝入潔凈
容器,一份作為試樣,一份作為留樣,密封并做好標(biāo)識(shí),于-18 ℃下保存。
8 測(cè)定步驟
8.1 提取
稱取約5 g試樣(精確到0.01 g)于50 mL離心管中,加入乙腈(5.1)10 mL、水10 mL,渦旋混勻
2 min后,加入無(wú)水硫酸鎂(5.5)4.5 g、氯化鈉(5.6)1 g、檸檬酸鈉(5.7)0.3 g、檸檬酸氫二鈉(5.8)
0.7 g,渦旋混合器中振搖25 min,4 000 r/min離心5 min,待凈化。
8.2 凈化
取1 mL上清液(見(jiàn)8.1)于裝有100 mg C18(5.16)的2 mL離心管中,渦旋搖勻2 min,10 000 r/
min離心5 min,取上清液過(guò)濾膜(5.17),供高效液相色譜儀測(cè)定。
8.3 測(cè)定
8.3.1 液相色譜條件
由于測(cè)試結(jié)果取決于所使用的儀器,因此不可能給出液相色譜分析的通用參數(shù)。設(shè)定的參數(shù)應(yīng)保
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證色譜分析時(shí)被測(cè)組分與其他組分能得到有效的分離,下列給出的參數(shù)可供參考。
液相色譜參考條件如下:
a) 色譜柱:C18 柱,100 mm ×2.1 mm(內(nèi)徑),粒度3.5 μm 或性能相當(dāng)者;
b) 流動(dòng)相:0.1%磷酸水溶液和乙腈,梯度洗脫,洗脫程序見(jiàn)表1;
c) 流速:0.5 mL/min;
d) 進(jìn)樣量:10 μL;
e) 柱溫:35 ℃;
f) 檢測(cè)波長(zhǎng):松香酸240 nm,脫氫松香酸210 nm。
表1 梯度洗脫程序
時(shí)間/min A/%(0.1%磷酸水溶液) B/%(乙腈)
0 70 30
4 70 30
15 10 90
17 10 90
17.5 70 30
20 70 30
8.3.2 標(biāo)準(zhǔn)工作曲線的繪制
按8.3.1的測(cè)定條件,將標(biāo)準(zhǔn)工作溶液濃度由低到高依次進(jìn)樣測(cè)定,以目標(biāo)物色譜峰的峰面積為縱
坐標(biāo),與其對(duì)應(yīng)的溶液濃度為橫坐標(biāo)作圖,繪制標(biāo)準(zhǔn)工作曲線。高效液相色譜圖見(jiàn)附錄B中的圖B.1
和圖B.2。
8.3.3 試樣測(cè)定
按8.3.1的條件測(cè)定樣品和標(biāo)準(zhǔn)工作溶液,如果檢測(cè)的松香酸和脫氫松香酸的色譜峰保留時(shí)間與
標(biāo)準(zhǔn)品保留時(shí)間相差不超過(guò)±2.5 %,則可初步確認(rèn)樣品中存在松香酸和脫氫松香酸。必要時(shí),陽(yáng)性樣
品需用液相色譜-質(zhì)譜/質(zhì)譜法進(jìn)行確認(rèn)試驗(yàn)(見(jiàn)附錄C),松香酸或脫氫松香酸定性離子對(duì)的相對(duì)豐度
(是用相對(duì)于最強(qiáng)離子豐度的強(qiáng)度百分比表示)與濃度相當(dāng)基質(zhì)標(biāo)準(zhǔn)工作溶液的相對(duì)豐度一致,相對(duì)豐
度允許偏差不超過(guò)表C.2規(guī)定的范圍,且每個(gè)定性離子的信噪比均≥ 3,則可判斷樣品中存在松香酸和
脫氫松香酸。松香酸和脫氫松香酸的多反應(yīng)監(jiān)測(cè)色譜圖見(jiàn)附錄D 中的圖D.1和圖D.2。選取響應(yīng)值
相近的基質(zhì)混合標(biāo)準(zhǔn)工作液一起進(jìn)行色譜分析,基質(zhì)混合標(biāo)準(zhǔn)工作液和待測(cè)液中松香酸和脫氫松香酸
的響應(yīng)值在儀器線性范圍內(nèi),超過(guò)線性范圍則應(yīng)稀釋后再進(jìn)樣分析。外標(biāo)法定量。
8.4 空白試驗(yàn)
試劑空白試驗(yàn):除不加試樣外,均按上述步驟進(jìn)行。
試樣空白試驗(yàn):取空白基質(zhì)試樣,除不加標(biāo)準(zhǔn)溶液外,均按上述步驟進(jìn)行。
9 計(jì)算結(jié)果與表述
試樣中松香酸或脫氫松香酸含量由色譜數(shù)據(jù)處理軟件或按公式(1)計(jì)算獲得,計(jì)算結(jié)果應(yīng)扣除空
白值。
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X =C×V×1 000 m ×1 000 ………………………………( 1 )
式中:
X ———試樣中松香酸或脫氫松香酸含量的數(shù)值,單位為毫克每千克(mg/kg);
C———從標(biāo)準(zhǔn)工作曲線得到的松香酸或脫氫松香酸濃度的數(shù)值,單位為微克每升(μg/L);
V———試樣溶液最終定容體積的數(shù)值,單位為毫升(mL);
m ———最終定容體積試樣溶液所代表試樣質(zhì)量的數(shù)值,單位為克(g)。
計(jì)算結(jié)果保留3位有效數(shù)字。
10 檢出限、定量限、回收率和精密度
10.1 檢出限和定量限
本方法松香酸和脫氫松香酸的檢出限均為0.20 mg/kg,定量限均為 0.50 mg/kg。
10.2 回收率和精密度
本方法分別以雞肉、鴨肉、鵝肉、羊肉、豬肉、牛肉為空白樣品基質(zhì),進(jìn)行三個(gè)濃度水平的添加回收試
驗(yàn),每個(gè)濃度水平進(jìn)行6次重復(fù)實(shí)驗(yàn),測(cè)得各種基質(zhì)中松香酸和脫氫松香酸的回收率范圍與精密度,見(jiàn)
附錄E中的表E.1。
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附 錄 A
(資料性)
松香酸和脫氫松香酸標(biāo)準(zhǔn)物質(zhì)的信息
松香酸和脫氫松香酸的中文名稱、英文名稱、CAS號(hào)、分子式、結(jié)構(gòu)式和相對(duì)分子質(zhì)量見(jiàn)表A.1。
表A.1 松香酸和脫氫松香酸標(biāo)準(zhǔn)物質(zhì)的信息
中文名稱英文名稱CAS號(hào)分子式結(jié)構(gòu)式相對(duì)分子質(zhì)量
松香酸Abietic acid 514-10-3 C20H30O2 302.46
脫氫松香酸Dehydroabietic acid 1740-19-8 C20H28O2 300.44
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附 錄 B
(資料性)
松香酸和脫氫松香酸標(biāo)準(zhǔn)溶液的高效液相色譜圖
松香酸和脫氫松香酸標(biāo)準(zhǔn)溶液的高效液相色譜圖見(jiàn)圖B.1與圖B.2。
圖B.1 松香酸標(biāo)準(zhǔn)溶液的高效液相色譜圖(2.50 mg/L)
圖B.2 脫氫松香酸標(biāo)準(zhǔn)溶液的高效液相色譜圖(2.50 mg/L)
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附 錄 C
(資料性)
液相色譜-質(zhì)譜/質(zhì)譜法確證條件1)
1) 非商業(yè)性聲明:附錄C所列參考質(zhì)譜條件是在 AB 4000Q trap型液質(zhì)聯(lián)用儀上完成的,此處列出試驗(yàn)用儀器型
號(hào)僅為提供參考,并不涉及商業(yè)目的,鼓勵(lì)標(biāo)準(zhǔn)使用者嘗試不同廠家或型號(hào)的儀器。
C.1 液相色譜條件
液相色譜條件如下:
a) 色譜柱:C18 柱,100 mm ×2.1 mm(內(nèi)徑),3.5 μm,或性能相當(dāng)者;
b) 流動(dòng)相:0.1%甲酸-乙腈(等度洗脫8 min,15∶85,體積比);
c) 流速:0.5 mL/min;
d) 柱溫:35 ℃;
e) 進(jìn)樣量:10 μL。
C.2 質(zhì)譜參考條件
質(zhì)譜參考條件如下:
a) 電噴霧電壓:5 000 V;
b) 離子源溫度:550 ℃;
c) 氣簾氣:25 psi;
d) 霧化氣:50 psi ;
e) 加熱輔助氣:50 psi ;
f) 松香酸和脫氫松香酸的主要質(zhì)譜參考參數(shù)見(jiàn)表C.1,定性確證時(shí)相對(duì)離子豐度的最大允許偏
差見(jiàn)表C.2。
表C.1 松香酸和脫氫松香酸的主要質(zhì)譜參考參數(shù)
化合物電離模式
母離子
m/z
子離子
m/z
去簇電壓
V
碰撞能量
eV
駐留時(shí)間
s
松香酸ESI+ 303.2 257.3 70.0 19.3 0.2
123.1* 70.0 21.7 0.2
脫氫松香酸ESI+ 301.0 173.2* 89.4 19.0 0.2
159.2 89.4 24.1 0.2
注: 帶“*”的離子為定量離子。
表C.2 定性確證時(shí)相對(duì)離子豐度的最大允許偏差
相對(duì)離子豐度(基峰)/% >50 >20~50(含) >10~20(含) ≤10
允許的相對(duì)偏差/% ±20 ±25 ±30 ±50
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附 錄 D
(資料性)
松香酸和脫氫松香酸標(biāo)準(zhǔn)溶液的監(jiān)測(cè)色譜圖
松香酸和脫氫松香酸標(biāo)準(zhǔn)溶液的監(jiān)測(cè)色譜圖見(jiàn)圖D.1和圖D.2。
a) 松香酸標(biāo)準(zhǔn)溶液定性離子色譜圖
b) 松香酸標(biāo)準(zhǔn)溶液定量離子色譜圖
圖D.1 松香酸標(biāo)準(zhǔn)溶液的監(jiān)測(cè)色譜圖(0.50 mg/L)
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a) 脫氫松香酸標(biāo)準(zhǔn)溶液定量離子色譜圖
b) 脫氫松香酸標(biāo)準(zhǔn)溶液定性離子色譜圖
圖D.2 脫氫松香酸標(biāo)準(zhǔn)溶液的監(jiān)測(cè)色譜圖(0.50 mg/L)
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附 錄 E
(資料性)
畜禽肉中松香酸和脫氫松香酸的添加回收率和精密度(n=6)
畜禽肉中松香酸和脫氫松香酸的添加回收率和精密度(n=6)見(jiàn)表E.1.
表E.1 畜禽肉中松香酸和脫氫松香酸的添加回收率和精密度(n=6)
基質(zhì)添加濃度/(mg/kg)
回收率范圍/% RSD/%
松香酸脫氫松香酸松香酸脫氫松香酸
雞肉
0.500 84.4~99.6 104~107 6.61 1.34
1.00 94.0~106 96.2~108 4.06 4.55
5.00 89.4~95.2 87.6~90.6 2.21 1.56
鴨肉
0.500 98.0~101 96.0~102 1.16 2.01
1.00 86.2~107 86.0~97.6 7.47 5.21
5.00 83.6~91.8 86.9~89.4 3.81 1.20
鵝肉
0.500 90.8~109 91.2~104 8.00 5.66
1.00 94.0~106 88.6~97.0 5.14 3.40
5.00 88.8~93.0 89.0~95.2 2.23 2.48
豬肉
0.500 93.2~102 87.2~98.0 3.71 4.38
1.00 93.6~106 92.6~106 5.12 5.01
5.00 90.0~94.4 85.4~90.6 1.70 2.28
牛肉
0.500 96.4~100 88.0~98.8 1.37 4.26
1.00 84.6~92.4 88.8~90.8 3.52 0.73
5.00 85.0~93.6 90.2~98.8 3.24 4.17
羊肉
0.500 90.0~106 100~104 7.90 1.78
1.00 96.4~110 99.6~104 5.73 1.83
5.00 86.6~94.2 89.6~94.0 3.05 1.95
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SN/T5639—2024
Foreword
This standard was drafted according to GB/T 1.1—2020.
Please note that some of the contents of this document may involve patents. The publisher of this
document does not assume the responsibility of identifying these patents.
This standard was proposed by and under the charge of General Administration of Customs of the
Pepople’s Republic of china.
This standard was drafted by GuangZhou Customs District P. R. China, China Meat Research Center,
Science and Technology Research Center of China Customs.
This standard was mainly drafted by Li Ju, Xie Jian Jun, Zeng Guang Feng,Gu Jin, Wang Lu,Han
Shen,Xi Jing, Dong Jie, Chen Wen Rui, Wang Zhi Yuan, Hou Ying Ye.
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Determination of abietic acid and dehydroabietic acid
in livestock and poultry muscles for export
1 Scope
This standard specifies the method of determination of abietic acid and dehydroabietic acid in livestock
and poultry muscles for export by HPLC, and confirmation by LC-MS/MS.
This standard is applicable to the determination and confirmation of abietic acid and dehydroabietic
acid in animal origin foodstuffs shuch as chicken, duck, goose meat, pork, beef, mutton etc.
2 Normative reference
The following referenced documents are indispensable for the application of this document.
For dated references, only the edition cited applies.For undated references, the latest edition of the
referenced document (including any amendments) applies.
GB/T 6682 water for analytical laboratory use—Specification and test method
3 Terms and definitions
There is no term or definition to be defined in this document.
4 Principle
The abietic acid and dehydroabietic acid in the test sample is extracted with acetonitrile , thereafter
being cleaned up by C18 purifying agent bonded silica. the contents are determined by HPLC with PDA
(photo-diode array) or UV detector, quantifired by external standard method, and the positive sample
is qualitatively confirmed by LC-MS/MS.
5 Reagent and materials
Unless otherwise specified, all regents used should be of analytical reagent(AR), “water” is the first
grade water prescribed by GB/T 6682.
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5.1 Acetonitrile: Chromatogram grade.
5.2 Methanol: Chromatogram grade.
5.3 Formic acid:Chromatogram grade.
5.4 Phosphoric acid: Guaranteed reagent.
5.5 Magnesium sulfate anhydrous.
5.6 Sodium chloride.
5.7 Trisodium citrate.
5.8 Disodium hydrogen citrate sesquihydrate.
5.9 0.1% Phosphoric acid solution: accurately pipette 1 mL phosphoric acid(5.4) dilute to 1000
mL with water,mix to homogenous.
5.10 Abietic acid: purity(ca 98.0%),the information of abietic acid is listed in Table A.1 in
Annex A.
5.11 Dehydroabietic acid: purity(ca 98.0%),the information of dehydroabietic acid is listed in Table
A.1 in Annex A.
5.12 Abietic acid standard stock solution(1 000 mg/L): Accurately weigh 10 mg of abietic acid
standard (5.10) in a small beaker, dissolve with a small amount methanol(5.2), to scale in the
10 mL volumetric flask then make up to graduation with Methanol, shake. the standard stock solution
should be kept away from light and stored at -18 ℃. The term of validity is 6 months.
5.13 Dehydroabietic acid standard stock solution(1 000 mg/L): Accurately weigh 10 mg of dehydroabietic
acid standard (5.11) in a small beaker, dissolve with a small amount Methanol, to scale
in the 10 mL volumetric flask then make up to graduation with Methanol, shake. the standard stock
solution should be kept away from light and stored at -18 ℃. The term of validity is 6 months.
5.14 Mixed standard intermediates solution(100 μg/mL): Accurately transfer 1.00 mL standard
stock solution(5.12, 5.13)into 10 mL volumetric flask, dilute to the scale with acetonitrile(5.1).
should be kept away from light and stored at -18 ℃. The term of validity is 1 month.
5.15 Mixed matrix standard working solution: Dilute the mixed standard intermediate solution
(5.14) with acetonitrile(5.1), a series of concentration of the standard solution : 0.10 mg/L 、0.20
mg/L 、0.50 mg/L、1.00 mg/L、2.00 mg/L and 5.00 mg/L, Prepare just before use.
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5.16 Silica Gel bonded C18 purifying agent:particle size 50 μm.
5.17 Membrane filter: 0.22 μm, organic type.
6 Apparatus and equipment
6.1 High performance liquid chromatograph : equipped with PDA or UV detector.
6.2 High performance liquid chromatography tandem mass spectrometry: equipped with electrospray
ionization source (ESI).
6.3 Analytical balance: capable of accurately weighing to 0.01 g and 0.1 mg.
6.4 Tissue blender.
6.5 Vortex mixer.
6.6 Centrifuge: not less than 4 000 r/min.
6.7 High speed centrifuge: not less than 10 000 r/min.
6.8 Nitrogen evaporator.
7 Preparation and storage of test sample
7.1 In the course of sample preparation, precaution must be taken avoid the contamination or any
factors which may cause the charge of residue content.
7.2 Take a representative sample(livestock meat samples with skin) of about 500 g , grind and and
homogenize, put into a clean container,divide into two groups, with one part as the sample and the
other as the retention sample. Seal and make labeling. stored at -18 ℃.
8 Procedure
8.1 Extraction
Weigh 5.0 g (accurate 0.01 g) of the test sample into 50 mL centrifuge tube. Add 10 mL acetonitrile
(5.1) and 10 mL water, blend with cap, Vortex for 2 min, then add 4.5 g Magnesium sulfate anhydrous(
5.5), 1 g Sodium chloride(5.6), 0.3 g Trisodium citrate(5.7), 0.7 g Disodium hydrogen citrate
sesquihydrate(5.8), oscillate for 25 min, centrifuge at 4 000 r/min for 5 min after well blend,
for clean up.
8.2 Clean up
Transfer 1 mL the supernatant(8.1) into a 2 mL centrifuge tube which had 100 mg C18(5.16). Vortex
for 2 min, centrifuge at 10 000 r/min for 5 min, then filter the supernatant through 0.22 μm Membrane
filter(5.17) and ready for analysis by HPLC.
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8.3 Determination
8.3.1 Liquid chromatography conditions
As the test result depends on test condition of instrument, so it is not possible to take the general
parameters for the LC chromatography , the parameters should be able to identify the components of
the test group and the other components can be separated effectively, the following parameters can
be used for reference:
HPLC operation conditions are as follows:
a) LC column: C18 LC column (100 mm ×2.1 mm id .), 3.5 μm or equivalent;
b) Mobile phase:A is Phosphoric acid (5.3),B is acetonitrile(5.1),the grade of mobile phase is listed
in table 1.
c) Flow rate: 0.5 mL/min.
d) Injection volumn: 10 μL.
e) Column temperature: 35 ℃.
f) Wave length: abietic acid is 240 nm, dehydroabietic acid is 210 nm.
Table 1—Grade of mobile phase
Time/min A/%(0.1% Phosphoric acid solution) B/%(Acetonitrile)
0 70 30
4 70 30
15 10 90
17 10 90
17.5 70 30
20 70 30
8.3.2 Draw curve of the working solution curve
Base on the determination conditions of 8.3.1, the standard working solution was injected from low
to high, and the chromatographic peak area is taken as the ordinate, and the corresponding solution
concentration is taken as abscissa, draw curve of the standard working solution. The liquid chromatography
of them are shown in figures B.1 and figures B.2 in Annex B.
8.3.3 Qualitation and quantification determination of sample
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Determine the solution of test sample and the standard working solution according to the determination
conditions in 8.3.1.If the retention time of the chromatographic peak of abietic acid and dehydroabietic
acid from the test sample is less than ±2.5% compared with the standard solution, the
presence of abietic acid and dehydroabietic acid in the test sample can be preliminarily confirmed. If
necessary, the positive sample should be qualitatively confirmed by LC-MS/MS (referenced Annex
C).The relative intensities (Expressed as a percentage of intensity relative to the strongest ion
abundance)of abietic acid and dehydroabietic acid from sample transitions shall correspond to those
of mixed matrix standard solution transitions for confirmation, the concentration of mixed matrix
standard solution should be same with those of sample solution. The deviation of the relative abundance
does not exceed the provisions in Table C.2 in Annex C, and the S/N ≥3,it can be judged that
there are abietic acid and dehydroabietic acid in the sample. The multiple reaction monitoring (MRM)
chromatograms of abietic acid and dehydroabietic acid are shown in figures D.1 and D.2 in Annex
D. Select the mixed matrix standard working solution with similar response value for chromatographic
analysis ,the responses of abietic acid and dehydroabietic acid in the mixed matrix standard
working solution should be in the linear range of the instrumental detection, if the concentration of
the test sample exceed the line range, the test sample should be diluted before injection analysis again.
And quantifired by external standard method.
8.4 Blank test
Solvent blank: The operation of the blank test is the same as that described in the method of determination,
but with the omission of sample addition.
Sample blank: The operation of the sample blank test is the same as that described in the method of
determination, but with the omission of abietic acid and dehydroabietic acid addition.
9 Calculation and expression of the result
Calculate the content of abietic acid and dehydroabietic acid in the test sample by LC data processor
or according to the followed formula(1), the blank values should be deducted from the calculation
result:
X=C×V×1 000
m ×1 000
…………………………( 1 )
Where:
X———the content of abietic acid or dehydroabietic acid in the test sample, mg/kg;
C———the concentration of abietic acid or dehydroabietic acid of test sample in the standard
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working solution,
g/L;
V———the final volume of the test sample solution, mL;
m ———the mass of the test sample in final sample solution, g.
three significant figure should be retained.
10 Limit of detection(LOD),limit of quantitation(LOQ) ,recoveries and accuracy
10.1 LOD and LOQ
The LOD of this method for abietic acid or dehydroabietic acid is 0.20 mg/kg, the LOQ for abietic
acid or dehydroabietic acid is 0.50 mg/kg.
10.2 Recoveries and accuracy
This method was carried out in an indoor recovery experiment with different samples (chicken,
duck, goose meat, pork, beef, mutton) as a sample blank for three concentration levels of additive
recovery experiments. Each concentration level conducting 6 times repeated experiments. The
measured range of abietic acid and dehydroabietic acid recovery in Various matrix are shown in table
E.1 in Annex E.
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Annex A
(informative)
The compound information of abietic acid and dehydroabietic acid
The compound name, CAS No., Molecular formula, Structural formula and Molecular
weight of abietic acid and dehydroabietic acid are shown in table A.1.
Table A.1—The compound information of abietic acid and dehydroabietic acid
compound name CAS No. Molecular formula Structural formula Molecular weight
Abietic acid 514-10-3 C20H30O2 302.46
Dehydroabietic acid 1740-19-8 C20H28O2 300.44
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Annex B
(informative)
The liquid chromatograms of abietic acid and dehydroabietic acid standard solution
The liquid chromatography of abietic acid and dehydroabietic acid starndard solution are shown in
figures B.1 and B.2 in Appendix B.
Figure B.1—Liquid chromatograms of abietic acid standard solution(2.50 mg/L)
Figure B.2—Liquid chromatograms of dehydroabietic acid standard solution(2.50 mg/L)
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Annex C
(informative)
LC-MS/MS operating condition1)
1) Non-commercial statement: Referenced conditions in Annex C were obtained from AB 4000Q trap LC-MS/MS.
The equipments and their types involved in the standard method are not related to commercial aims, and it is
encouraged to use equipments of different corporation of different type.
C.1 LC operating conditions
LC operating conditions is flowing:
a) LC column: C18 LC column (100 mm ×2.1 mm id.),3.5 μm, or equivalent.
b) Mobile phase: 0.1% formic acid-acetonitrile(isogradient elution 8 min, 15∶85,V/V).
c) Flow rate: 0.5 mL/min.
d) Column temperature: 35 ℃.
e) Injection volumn: 10 μL.
C.2 MS/MS operating conditions
MS/MS operating conditions is flowing:
a) Capillary voltages :5 000 V.
b) Source temperature :550 ℃.
c) Curtain Gas pressure: 25 psi.
d) Nebulizer Gas pressure: 50 psi.
e) Aux Gas pressure: 50 psi.
f) Mass spectrometry parameters of abietic acid and dehydroabietic acid are shown in table C.1,
the maximum allowable deviation of relative ion abundance are shown in table C.2.
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Table C.1—Mass spectrometry parameters of abietic acid and dehydroabietic acid
compound name
Scanning
model
Parent ion
(m/z)
Danghter ion
(m/z)
de clustering
voltage/V
collision
energy/eV
dwell time/
s
abietic acid ESI+ 303.2
257.3 70.0 19.3 0.2
123.1* 70.0 21.7 0.2
dehydroabietic acid ESI+ 301.0
173.2* 89.4 19.0 0.2
159.2 89.4 24.1 0.2
Note: “*”represents the quantitative ion.
Table C.2—The maximum allowable deviation of relative ion abundance
Relative ion abundance(base peak)/% >50 >20~50 >10~20 ≤10
Allowable relative deviation/% ±20 ±25 ±30 ±50
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Annex D
(informative)
The MRM chromatograms of abietic acid and dehydroabietic acid standard solution
The MRM chromatograms of abietic acid and dehydroabietic acid standard solution are shown in figures
D.1 and figures D.2
a) Qualitative ion chromatogram of abietic acid standard solution
b) Quantitative ion chromatogram of abietic acid standard solution
Table D.1—The MRM chromatograms of abietic acid standard solution(0.50 mg/L)
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a) Quantitative ion chromatogram of dehydroabietic acid standard solution
b) Qualitative ion chromatogram of dehydroabietic acid standard solution
Table D.2—The MRM chromatograms of dehydroabietic acid standard solution(0.50 mg/L)
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Annex E
(informative)
Range of recovery and RSDs for abietic acid and dehydroabietic
acid in livestock and poultry muscles(n=6)
Range of recovery and RSDs for abietic acid and dehydroabietic acid in livestock and poultry muscles
are shown in table E.1.
Table E.1—Range of recovery and RSDs for abietic acid and dehydroabietic acid
in livestock and poultry muscles(n=6)
Matrix Spiked level/(mg/kg)
Range of recovery/% RSD/%
abietic acid dehydroabietic acid abietic acid dehydroabietic acid
chicken
0.500 84.4~99.6 104~107 6.61 1.34
1.00 94.0~106 96.2~108 4.06 4.55
5.00 89.4~95.2 87.6~90.6 2.21 1.56
duck
0.500 98.0~101 96.0~102 1.16 2.01
1.00 86.2~107 86.0~97.6 7.47 5.21
5.00 83.6~91.8 86.9~89.4 3.81 1.20
goose meat
0.500 90.8~109 91.2~104 8.00 5.66
1.00 94.0~106 88.6~97.0 5.14 3.40
5.00 88.8~93.0 89.0~95.2 2.23 2.48
pork
0.500 93.2~102 87.2~98.0 3.71 4.38
1.00 93.6~106 92.6~106 5.12 5.01
5.00 90.0~94.4 85.4~90.6 1.70 2.28
beef
0.500 96.4~100 88.0~98.8 1.37 4.26
1.00 84.6~92.4 88.8~90.8 3.52 0.73
5.00 85.0~93.6 90.2~98.8 3.24 4.17
mutton
0.500 90.0~106 100~104 7.90 1.78
1.00 96.4~110 99.6~104 5.73 1.83
5.00 86.6~94.2 89.6~94.0 3.05 1.95
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