【商检行业标准(SN)】 进出口保健食品中伐地那非、西地那非、他达那非的检测方法 液相色谱-质谱/质谱法

中华人民共和国出入境检验检疫行业标准SN/T 1951—2007
进出口保健食品中伐地那非、西地那非、他达那非的检测方法
液相色谱-质谱/质谱法
Determination of vardenafil, sildenafil and tadalafil in healthfoods Tor import and export--LC-MS/MS method2007-08-06发布
数前肤伤
中华人民共和国
国家质量监督检验检疫总周
2008-03-01实施
本标泄的附录A和降录1为资料附录。本标准出国家认证认可监督管理委员会提出并归口。本标准由中华人民共和国湖南出人境检验检疫局起草本标上要起草夫：王美玲、万向阳、戴华、李拥军、主正良、工象贤、黄葬。本标批系首次发布的出人境检验检疫行业标难。SN/T 1951—2007
1范围
进出口保健食品中伐地那非、西地那非、他达那非的检测方法
液相色谱-质谱/质谱法
SN/T 1951—2007
本标准规定了保健食晶中找地那非凹地那非，恺达那非的液相色谱-质谱/质谱检测方法，本标准适用丁片剂胶囊剂研科服泌剂类保健食缺中栈地那菲两地那非、他达那非的检测。2测定方法
2.1方法提要
片剂、胶囊剂试样小的伐地邯非、西地那非疝达郝非用划爵超声提取，提取液经稀释后过滤。口服溶没剂经直接稀释后过滤，用配有电喷系离呼源的滚扣包谱质谐/质谱仪进行测定、外标法定。2.2试剂和材料
所有试剂除特殊注明外，均为奇纯水为款蒸馏水2.2.1乙脯：效液相色谱级。
中醇：高效液相色谱级，
冰艺酸：优级纯。
战酸：优级纯，
2.2.50. 1 1mo1/I.盐酸+甲醇合液有+1,体积化）2.2.6
我地邮非（vardenafil,CA第号：224785-94-4.分子式:C2HN.O,S·HC1·3H.O)标准品:纯度大于等于99.5%。
，外子式aNaO.s标雅品：纯度大手饰于2.2.7西地那非（SildenafilGAs 13975±-83-2，99.5%。
2.2.8他达那非（tadalafil，CA学，115969-5，分子式：C，N）标摊品：纯度大丁等于99.5%。2.2.9标雅溶液：分别推确你教适摄化延那非，西地那套，他达班非标准品，用甲醇配制成浓度为10 μ/ml.的混合标备液，
℃下保存：想掘需要用.1mml红盐酸+甲醇混合液(2.2.5)将标，4g/ml，fong/ml.50ng/mL.1cgng/mL的混合标准工作雅储备液稀释成1 ng/mf.、2 g/L液。
2.3仪器和设备
2.3.1高效液相总谱-质谱/质谱仪，三重叫极杆质谱检测器，配电喷雾离了源（ESI)。2.3.2超声波清洗器。
2.3.3涡混匀器。
2.3.4电子人平：感量1tng.
2.3.5研。
2.4测定步骤
2.4.1试样的制备
2.4. 1.1 片剂
随机取同一抵号的供试品20片，研细为试样，1
SN/T 1951—2007
2.4,1.2硬胶囊
随机收同一批号的供试品20粒，倾出所有内容物，研绷为试样。2.4.1.3软胶囊
随机取同一批号的供试品20粒，将内容物全部挤到一离心管中，充分匀后，作为试样。2.4.1.4口服溶液剂
随机抽取间批号的供试品10支（瓶），取等量体积样品到同一容器中泥匀，作为试样。2.4.2提取
2.4.2.1片剂、硬胶囊、软胶戳试样称取1g试样(精确到0. 01g)于100 mL客量瓶中,加入 90 ml.中醇，盖1盖混匀,臀于超声波清洗器山超尚 1 h,冷划至密溢后用甲酶定容至刻度,混句]。取 l,0 ml于[0 ml.容量瓶中,用 0. 1 mol/L盐酸十甲醇握合液（2.2.5)定容后混，液过0.45让m微孔滤膜后·供液相色谱-压谱/质谱仪测定。2.4. 2. 2口肢溶滋剂
准确移取 1. 0 mI.试样可 10 mL 等鼠中,用0. 1 Imal/1. 盐酸十甲醇混合液(2. 2. 5)定籍,混列.溶液过0.15m微孔滤膜后，供液和色谱-质增/质谱仪测定，2.4.3测定
2.4.3.1液相色谱-质谱/质谱条件色谱柱：柱填料为十八烷基硅烷键合相的色谱柱，2.1X150mmL5u功或相当者：a)
流动机，梯度洗脱程序见表11
流速:0.20 ml./min;
杜温：40℃：
进样垫:10 μL;
离子源：电喷离子源：
打描方式：止离子：
检测方式：多反应监测（MRM)：等化气、经气、辅助加热气、碰撞气等气体使用前应调出各气体流展以使质谱获敏度达到检i）
测要求，参考条件参见附录A
喷穿电压，去集获电压，碰撞能等心压值应优化至鼓优灵数度，参者条件参见附录A；监测离了对（m/2）：他达那非390.4/268.3（定量离子对）390.4/133.1、390、4/169，1断地那k）
非475.3/100.1(定最离子对)475.3/58,0,475.3/311.4,俊地那非189.5/151.1(定量离子对)498.5/72.1.489.5/312,1。
表1流动相梯度洗脱程序
时间/in
2.4.3.2液相色谱-质谱/质谱测定0.1%乙酸水溶液/%
乙隋/%
按照2.4.3.1液相色谱-质谱/质谱条性测定样液和标准工作济液，标准的线法测定样液中的伐地那非，西地那非、他达那非含。样液中鼓测物的响位值应代仪器线性范时之内，妇果超出仪器线性范SN/T 1951--2007
围，应用0.1101/L盐酸十比醇混合液（2.2.5进行适当帮释。在上述色谱条件下，伐地那非，西地那非，他达那非的质量色谱峰保留时间约为10.5min，10.7min，12.4min。标准溶液的提取离子流色谱图参见附录 3 中的图 3. 1。
在相同的实验条件下，样液中被测物的质摄色谱峰保留时间与标准工作溶液一致,并且在扣除背景后的样品包谱图中.所选择的离子对均出现，各延性阅子的相对丰度与浓度相当的标推溶液的离了机对丰度-致，误不超过表2规定的范同，则可判断样品中存在对应的被测物。表2定性确证时相对离子丰度的最大允许误差和对离了度/%
>20 ~·50
10~-20Www.bzxZ.net
2. 4. 3. 3空白试验
除不切试详外，均按上述测定步骤进行。2.5结果计算和表
允许的相对误差/%
用液机色谱-质谱/质数据处理机或按照式(1)计算样品中伐地那非、西地那非、他达那非含量，计筛结果弱扣除空白。
式中：
X-一试样中伐地那非、西地那非、他达部非的含最，单位为慈克千克（mg/kg）或者毫克每(mg/1.) :
样液中伐地哪非、西地那非，他达那非的仙积或峰高标推工作液中伐地娜非、西地那非他达那非的浓度，单位为微克每毫升（产/ⅡL）；As
标准工作液中伐地那非，西地那非、他达那非的峰面积或峰高；V.样液最终定容怀积，单位为毫升(mL)：“空白实验液中伐地那非.两地邯非、他达那非的峰面积或峰高：A.
一最终样没所代表的试样质量，单位为克（名）成者所代表的试样休积单位为宽升（mL）。3测定低限、回收率
3. 1测定低限
本力法片剂，胶衰剂样品中伐地那非、随那推、他达那非的测定低限均为1.00/kg测定口服落毅剂样品中伐地那非西地那非、他那非的测定低限均为，010 ng/。3.2回收率
3.2.1保健食品中伐地那非添加浓度及其回收率数据片剂、胶囊剂添加浓度在1.0ng/kg～10mg/kg时，回收率在89.3%~104%之间。口服溶液添加浓度在0.010tmg/1.~0.10mg/1.时，回收率在94.5%~114%之间。3.2.2保健食品中西地那非添加浓度及其回收率数据片剂胶套剂添加依度在1.0mg/kg~10mR/kg时，回收率在83.8%~101%之间，几服浮液添加浓度在0.010r1g/1.~0.10mg/1时，向收率在85.1%～108%之间。3.2.3保健食品中他达那非添加浓度及其回收率数据片剂、胶囊剂添加浓度在1.0mg/kg～10 mg/kg时，回收率在81.5%~108%之间。口服落液添加浓度在0.010m%/L～0.10mg/L时，回收率在85.7%~100%之间。3
SN/T 19512007
附录A
（资料性附录）
API4000LC-MS/MS系统检测伐地那非，西地那非，他达那非参考条件1AP14000I.C-MS/MS系统电喷雾离子源参考条件：a)
窗帘气(CR),20.00 Psi;
化气（s1)：40.00Psi：
辅助加热气(GS2):45.00 Psi:
碰擅气(CAD):7.00 Psi;
离子源喷雾器电压（IS)：5行0360曦等针温度（TEM）：550.0
定性离子对、定基离子对去族电F、碰挖能摄、疆撞室出电压见表A，1。表A.13种待测物定性离子对、定离字对去簇电压、碰撞能置和碰撞案出口电压特汇物
伐地非
西地那非
拖达哪非
对定量离手对，
100rnfa
共蒙电
.79.00
碰控能基/V
链撞室出口电压/V
1）非商业性声明：本标准所来用仪器设备及型号不淤及商业目的，鼓励标准使用者尝试不同厂家或型号的仪器。1. &e5
附录B
（资料性附录）
伐地那非，西地那非、他达那非混会标准溶液提取离子流色谱图mtz 489. 5/151.1
m/z 475. 3/100. 1
mlz390.4/268.3
伐般那非
西璃洲生
恒达邪羊
SN/T1951-2007
伐地那非、西地那非、他达那非滬合标准溶液提取离子流色谱图图 B. 1
52/T1951—2007
Foreword
AnnexAand annexBofthis standardare infarmativeannexes.This standard was proposed by and is under the charge of the Certification and Accreditation Admin-istration of the People's Republic of China.This standard was drafted by the Hunan Entry-Exit Inspection and Quarantine Brureau of the People'sRepublic ot China.
The standard was mainly dratted by Wang Meiling. Wan Xiangyang, Dai Hua, Li Yongjun, WangZhengtiang, Wang Xiangxiar and Huang Ping.This standard is a professional standard for entry-exit inspection and quarantine promulgated for thefirst tirme.
Nore: This English version.a transtation from the Chinese text, is solely for guidance,SN/T 1951—2007
Determnination of vardenafil, sildenafil and tadalafil in healthfoods for import and export--Lc-Ms/Ms method1Scope
This staridard specifies the determination of vardenafil, sildenafil and tadalafil in health foods by LC-MS/MS method.
This standard is applicable to the determination of vardenafil, sildenafil and tadalafil in health foodsfor troche, capsule, soft capsule and oral solution.2 Methad of deterrnination
2. 1 Abstract of method
vardenafil, sildenafil and tadalafil in the troche, capsule and soft capsule samples are axtracted withmethanol in a ultrasonic washer and then the extract is diluted and filtered off. The oral solution sample is only diluted and filtered off, The diluted solution is determined by Lc-Ms/Ms, using exter-nal standard method.
2,2Reagents and materials
Unless otherwise specified, all the reagents should be of analytical grade and double-distilled water is used.2, 2. 1 Acetonitrile:HPLC grade.2.2.2 Methanol.HPLC grade.
2. 2. 3 Acetic acid,Guaranteed grade.2.2.4Hydrochloricacid:Guaranteed grade.2. 2. 5 0. 1 mol/1 hydrochloric acid solution-methanal (9 + 1, V/ V).2.2.6 Vardenafil (CAs No:224785-90-4. molecular formula:CHN.O,S -HCI -3H,O) standard:Purity99.5%
2. 2. 7 Sildenafil (CAS No: 139755-83-2, rnolecular formula:Ca HN.O, S)standard: Purity99. 5%7
SN/T1951—2007
2.2.8 Tadalafit (CAs No: 171596-29-5, molecular tormula.CH,N,0,> standard: Purity99. 5%2. 2. 9 Standard solution; accurately weight an adequate amount of vardenafil, sildenafil ard tadalatil stand-ard in an volumetric flask and dissolve with metharol to form a mixed standard stock solution of 10 μg/mlin concentration. and store at 4c. Dilute the mixed standard stack solution with 0. 1 mol/L mydrochloricacid solution-methanol (2, 2. 5) to obtain a series af the mixed standard working solutions with concentra-tions of1,2,4,10,50and100 ng/ml2.3 Apparatus and equipment
2.3.1High performance liquid chromatography triple-quadrupolel tandem mass spectrometerequippedwith electrosprayionizatin source(Esl)2.3.2 Ultrasonic washer.
2.3. 3Vortex mixer.
2.3.4 Electronic balance: readabilitymjigram.2.3.5 Mortar.
2.4 Procedure
2. 4. 1Preparatian ot test sampl2.4.1. 1 Troche
Twenty troches are sampled at re2, 4. 1.2 Capsule
fom'and ground tnto poyder which is used as test sample.Twenty pills are sampled at random and the materiafs in every capsule are spilled out and ground intopower which is used as test sample.2. 4. 1. 3Saft capsule
Twenty pills are sampled at rardam and the materials in every capsue are spilled into the sarne cen-trifuge tube and sufficiently mixed. The mixect sample is used as test sample.2. 4. 1. 4 Dral solution
Ten bottes of oral solution are sampled at random. Transfer the same volume of solution from every8
SN/T 1951—2007
bottle into the same container and sufficiently mixed. The mixed sample is used as test sample.2. 4.2Extractlon
2, 4, 2. 1 Troche, capsule and soft capsuleWeigh 1 g (accurate to Q. 01 g) of the test sample inta a 100 mL volumetric flask, add 90 mL metha-nol, put the stopper and extract in a uftrasanic washer for 1 h, Cool down to room temperature, di.lute to mark with methanol and mix the contents. Transfer 1. 0 mL of the extract into a 10 mL volu-metric flask and dilute to mark with 0., 1 mol/L hydrochloric acid aqueous solution-methano (2.2. 5).The diluted solution is fittered throtrghr-a d-45 --.2. 4. 2. 2 Oral solution
Accurately transfer 1. 0 ml of test sample intor?detarmined by Lc-MS/Ms.
10 mL volumietric flask and dilute to mark withD. 1 mof/L hydrochloric acid agueous solutron-methanot ,2. 2, 5). The diluted sofution is filteredthrough a o. 45 μm membrane and detetmined.by Lc,Ms/s.2.4. 3Determination
2.4. 3. 1 LC-MS/Ms operating cdnditionsa) Chromatographie column: octadecylsilica (s)or.oquivatenmcolumn(2.1x150mm,5μm)b) Mobile phase and gradient pfogram see tatfe 1Flowrate;0.20mL/min;
Columntemperature:4oc
e)Injection volume:10 μL:
f)lonsource:electrosprayionizationsource(Esi); Scan mode:positive ion;
Detection mode: multipte reaction monitoring (MRM) :i)Nebulizer Gas, curtain gas, collision gas and auxiliary gas should be optimized by adjusting thegas flow parameters.Raference conditions are shown in Annex A:j) lon spray voltage,daflector voltage, collision energy and sp on should be optimized to reach thehighest sensitive of mass spectrometer, Reterenced conditions are shown in Annex A:SN/T 1951—2007
Monitoring ions pairs (m/z):tadalafil 390.4/268.3 (quantitation ion pair),390.4/135,1,390.4/k)
169.1;sildenafil 475.3/100.1(guantitation ion pair),475.3/58.0,475.3/311.4,vardenafil489. 5/151. 1(quantitation ion pair), 498. 5/72. 1, 489. 5/312. 1.Table1-MobifephaseandgradientprogramTima/min
0. 1% Acetic acid agueous solution/%85.0
LC-MS/MS determination
2. 4. 3. 2
Acetonitrile:%
According to the Lc-Ms/Ms operating conditions (2. 4. 3. 1), the standard working solution and thesample solutian are determined, Quantitative analysis of the vardenafil, sildenafil and tadalafil in thesample solution is done using standard curve method. The responses of vardenafil, sildenafil andtadalafil in the sample solution shoufd be within the linear range of the instrumental detection, If theresponse is above the linear range.dilute the sample solution with O, 1 mol/L hydrochloric acid aqueous solution-methanal (2. 2. 5). Under the above chromatographic condition, the retention time ofvardenafil, sildenafil and tadalafil are ca 10. 5 min, 10. 7 min and 12. 4 min, respectively. Far Lc-Ms/Ms chromatogram of the standard, see Figure B. 1 in annex B,According to the Lc-Ms/Ms operating conditions ( 2. 4. 3. 1), if the retention time of sample chro-matogram peaks are consistent with the standards, and subtracted from background compensation:selected ions are all present and the relative ion ratio of the selected ions according with that of thecalibration standard. at comparable concentrations, within the tolerances (seen table 2), The corre.sponding analyte could be confirmed.Table 2--Maximum permitted tolerances for relative ion intensities while conflrmationRelative intensityr%
>20~50
2.4.3.3Blank test
Permitted tolerances/%
The operation of the blank test is the same as that described in the method of determination but10
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