《大蒜及其制品中蒜氨酸的測(cè)定 高效液相色譜法》編制說(shuō)明

1團(tuán)體標(biāo)準(zhǔn)《大蒜及其制品中蒜氨酸的測(cè)定高效液一、工作簡(jiǎn)況2022A03019-3）、新疆維吾爾自治區(qū)專利轉(zhuǎn)化項(xiàng)目（20230012）、2023大蒜(AlliumSativumL.)，是歷史悠久的藥食兩用植物。中國(guó)是世2藥典名稱質(zhì)控指標(biāo)備注美國(guó)藥典大蒜大蒜粉（凍干蒜粉）大蒜提取物粉大蒜流浸膏大蒜遲釋片蒜氨酸、L-γ-谷氨酰烯丙基半胱氨酸蒜氨酸、L-γ-谷氨酰烯丙基半胱氨酸蒜氨酸蒜氨酸蒜氨酸和潛在大蒜辣素蒜氨酸的檢測(cè)，樣品需要進(jìn)行衍生化，所用衍生化試劑叔丁基硫醇，臭鼬皮氣味，易燃、中毒，刺激性大，易造成環(huán)境污染應(yīng)為標(biāo)示量的90%-140%英國(guó)/歐盟藥典藥典大蒜潛在大蒜辣素蒜氨酸以羥苯丁酯為內(nèi)標(biāo)測(cè)定蒜氨酸的檢測(cè)，樣品在熱水中研碎，過(guò)濾鮮蒜中不存在的大蒜素。大蒜作為藥藥典大蒜大蒜素2010版、2015版、2020版《中國(guó)藥典》均規(guī)定大蒜的指標(biāo)成分為大蒜《中國(guó)藥典》1977年版收載的大蒜藥材標(biāo)準(zhǔn)中無(wú)含量測(cè)定項(xiàng)，2015前衍生化法，采用高效液相梯度洗脫，測(cè)定蒜氨酸（S-烯丙基-L-半胱氨酸亞砜，C6H11NO3S）的含量。缺點(diǎn)：該方法中的柱前衍生化用到了衍生化3辣素，屬于酶促反應(yīng)，受反應(yīng)體系中pH值、反應(yīng)溫度、底物量、底物濃序號(hào)標(biāo)準(zhǔn)號(hào)標(biāo)準(zhǔn)名稱適用范圍備注1NY/T1800-2009大蒜及制品中大蒜素的測(cè)定氣相色譜法蒜片、蒜油）中大蒜素類硫醚化合物（二烯丙基三硫醚、二烯丙基二硫醚）含量的測(cè)定大蒜素以二烯丙基三硫醚、二烯丙基二硫醚為代表，不能體現(xiàn)大蒜中的指標(biāo)性成分此測(cè)定指標(biāo)與美、英/歐盟各國(guó)對(duì)大蒜粉中質(zhì)控成分“潛在大蒜辣素”一2NY/T2643-2014大蒜及制品中高效液相色譜法蒜片）中蒜素的含量測(cè)定該標(biāo)準(zhǔn)采用了大蒜辣素（Allicin）為對(duì)照品，而大蒜辣素極其不穩(wěn)定，對(duì)照品需國(guó)外訂購(gòu)，其儲(chǔ)備液需-70℃以下冷凍保存，因此對(duì)測(cè)定結(jié)果帶來(lái)不確定性。此測(cè)定指標(biāo)與美國(guó)、印度藥典中對(duì)大3NY/T3871-2021大蒜中蒜氨酸的測(cè)定高效液相色譜法大蒜中蒜氨酸的含量測(cè)定蒜質(zhì)控成分“蒜氨酸”一致。該標(biāo)準(zhǔn)采用并優(yōu)化了印度藥典中大蒜的蒜氨酸含測(cè)方法，以蒜氨酸為對(duì)照品，以0.01%磷酸水溶液為流動(dòng)相，進(jìn)行含量測(cè)定4NY/T4351-2023大蒜及其制品中水溶性有機(jī)硫化合物的測(cè)定液相色譜-串聯(lián)質(zhì)譜法大蒜、蒜片及蒜粉中蒜氨酸、GSMC、GSAC、SMC、SAC、SPC的含量測(cè)定需用到配有電噴霧離子源的液相色譜-串聯(lián)質(zhì)譜儀，該儀器普及率很低且使用費(fèi)用高。4素標(biāo)準(zhǔn)品需國(guó)外訂購(gòu)，且不穩(wěn)定其儲(chǔ)備液需-70℃以下冷凍保存，因此對(duì)（3）中國(guó)農(nóng)業(yè)行業(yè)標(biāo)準(zhǔn)NY/T3871-2021《大蒜中蒜氨酸的測(cè)定高機(jī)硫化合物的測(cè)定液相色譜-串聯(lián)質(zhì)譜法》規(guī)定了大蒜及其制品中蒜氨酸等的含量測(cè)定，但需用到配有電噴霧離子源的液相色譜-串聯(lián)質(zhì)譜儀，（1）DB43/313-2006《茶陵紫皮大蒜》，由湖南省質(zhì)量技術(shù)監(jiān)督局T/HAS105-2020《飼料原料大蒜粉》，由河南省標(biāo)準(zhǔn)化協(xié)會(huì)發(fā)布。52023年3月，為了使標(biāo)準(zhǔn)更具代表性和適用性，《大蒜及其制品中2023年4月，本方法標(biāo)準(zhǔn)編制組按照項(xiàng)目組討論的意見(jiàn)，結(jié)合企業(yè)6大蒜藥用研究重點(diǎn)實(shí)驗(yàn)室、新疆天然藥物活性組分與釋藥技術(shù)重點(diǎn)實(shí)驗(yàn)7性別職務(wù)/職稱工作單位任務(wù)分工史榮梅女研發(fā)副總/高級(jí)實(shí)驗(yàn)師新疆胡蒜研究院（有限公司）標(biāo)準(zhǔn)起草劉睿婷女研發(fā)助理/高級(jí)實(shí)驗(yàn)師新疆大蒜藥用研究重點(diǎn)實(shí)驗(yàn)室標(biāo)準(zhǔn)起草宋百靈女研發(fā)助理新疆胡蒜研究院（有限公司）標(biāo)準(zhǔn)起草李新霞女副院長(zhǎng)/教授新疆醫(yī)科大學(xué)標(biāo)準(zhǔn)技術(shù)負(fù)責(zé)人馬雪紅女實(shí)驗(yàn)師新疆醫(yī)科大學(xué)樣品檢測(cè)李蘭蘭女/新疆醫(yī)科大學(xué)樣品檢測(cè)女總經(jīng)理新疆胡蒜研究院（有限公司）標(biāo)準(zhǔn)組織協(xié)調(diào)張春子女助理實(shí)驗(yàn)師新疆胡蒜研究院（有限公司）樣品檢測(cè)趙榮梅女新疆維吾爾自治區(qū)藥品檢驗(yàn)研究所大蒜產(chǎn)品信息胡小明男主任/正高自治區(qū)標(biāo)準(zhǔn)化研究院標(biāo)準(zhǔn)格式審核女主任/研究員中國(guó)科學(xué)院新疆理化技術(shù)研究所標(biāo)準(zhǔn)格式審核鄭瑞芳女主任/研究員新疆維吾爾自治區(qū)藥物研究所標(biāo)準(zhǔn)審核李莎莎女/新疆醫(yī)科大學(xué)樣品檢測(cè)女/新疆醫(yī)科大學(xué)樣品檢測(cè)），）；）），8），）；））美國(guó)藥典：AgilentZORBAXBonus-RP（4.6mm×250m）；9考察項(xiàng)目考察因素最佳條件測(cè)定方法美國(guó)藥典、印度藥典、自建方法3種方法無(wú)差異，自建方法前處理較簡(jiǎn)便滅酶時(shí)間5、10、15min10min）：定，置勻漿杯中，加入蒜酶抑制劑（羧甲基羥胺半鹽酸鹽溶液）70mL，）：）：min（20000r/min漿液定量轉(zhuǎn)移置500mL容量瓶中，加水稀釋至刻大蒜測(cè)定方法蒜氨酸含量（%）美國(guó)藥典印度藥典自建方法阿合奇大蒜木壘大蒜河南大蒜山東大蒜0.710.521.130.720.490.730.54滅酶時(shí)間（min）樣品編號(hào)稱樣量(g)蒜氨酸(%)平均含量(%)RSD(%)5123151.096150.204050.409850.27950.7380.7400.7290.7780.7360.7723150.743050.531250.73910.7560.7740.6920.7692350.244251.71900.6910.6840.6890.57搖勻，離心（8000r/min）15min，精密移取上清液1mL，置50mL量（5）大蒜粉（片）、大蒜片劑前處理考察考察項(xiàng)目考察因素最佳條件滅酶試劑甲醇、乙醇、羧甲基羥胺半鹽酸鹽溶液滅酶用量15、20、25mL20mL滅酶時(shí)間5、10、15min10min滅酶試劑樣品編號(hào)稱樣量(g)蒜氨酸(%)平均含量(%)RSD(%)羧甲基羥胺半鹽酸鹽溶液12310.30840.30410.30430.30723.413.423.463.883.430.892310.30770.30190.30533.823.893.863.860.90230.30720.30343.873.783.84滅酶用量（mL）樣品編號(hào)稱樣量(g)蒜氨酸(%)平均含量(%)RSD(%)10.30423.7420.30603.813.7630.30203.7310.30843.942020.30913.873.8830.30753.8210.30843.872520.30773.953.8830.30903.82滅酶試劑的用量為20、25mL時(shí)，蒜氨酸含量一致，故選擇甲醇用量為滅酶時(shí)間（min）樣品編號(hào)稱樣量(g)蒜氨酸(%)平均含量(%)RSD(%)512310.30540.30600.30410.30843.863.893.873.933.870.422310.30910.30750.30113.963.903.943.930.79230.30640.30573.933.933.930.19稱取蒜粉（片）、大蒜片劑0.3g（精確至0.0001g）置于50mL室溫冷卻，用水稀釋定容至刻度，混勻，離心考察項(xiàng)目考察因素最佳條件提取溫度45、55、55、60℃55℃提取方式磁力攪拌、超聲加熱、水浴攪拌磁力攪拌滅酶試劑甲醇、乙醇、羧甲基羥胺半鹽酸鹽溶液甲醇滅酶時(shí)間5、10、15、20min15min滅酶用量15、20、25mL15mL提取時(shí)間3、5、10min5min樣品名稱樣品編號(hào)稱樣量峰面積蒜氨酸(%)平均含量RSD(%)提取率10.29833849024.02大蒜粉20.29843818713.994.000.60/30.29973821823.98大蒜軟膏劑45℃1231.07001.08341.02882804142788542697190.4090.4010.4090.4061.0101.6大蒜軟膏劑50℃1231.07531.05471.04192652392683282634530.3850.3970.3940.3921.698.0大蒜軟膏劑55℃1231.04171.05031.08132773232823732867440.4150.4190.4130.4160.71104.0大蒜軟膏劑60℃1231.00261.01121.08382609342491492852140.4060.3840.4100.4003.5100.0由表可知，不同提取溫度對(duì)大蒜軟膏劑中蒜氨酸提取率有影響，在樣品名稱樣品編號(hào)稱樣量峰面積蒜氨酸(%)平均含量RSD(%)提取率磁力攪拌12311.04171.05031.08131.08502773232823732867442679570.4150.4190.4130.3920.4160.71104.0超聲加熱2311.07961.04901.09062685192569862807410.3950.3890.4090.3920.7698.1水浴攪拌231.06471.02502708282686140.4040.4160.410102.5蒜氨酸在磁力攪拌提取的方式下提取率為1滅酶劑名稱樣品編號(hào)稱樣量峰面積蒜氨酸平均含量RSD(%)提取率11.04172773230.41521.05032823730.4190.4160.71104.031.08132867440.41311.04392827850.422231.08281.02542854642685770.4110.4080.414103.5羧甲基羥胺11.07852813730.407半鹽酸鹽溶液231.08931.07702823742802180.4040.4060.4060.32101.4提取率分別為104.0%、103.5%、101.滅酶時(shí)間（min）樣品編號(hào)稱樣量峰面積蒜氨酸(%)平均含量RSD提取率512311.06931.04391.05711.07152793892719712765712672000.4070.4060.4080.3890.4070.21101.82311.07741.01861.04172875042719802773230.4160.4160.4150.4073.9101.82311.05031.08131.07012823732867442812480.4190.4130.4100.4160.71104.020231.06251.08872790442816300.4090.4030.4080.89101.9滅酶溶劑（mL）樣品編號(hào)稱樣量峰面積蒜氨酸(%)平均含量RSD(%)提取率12311.04171.05031.08131.07972773232823732867442881290.4150.4190.4130.4200.4160.71104.0202311.04451.04981.06152723542712822734550.4110.4070.4060.413103.225231.04711.06562746372822540.4130.4170.412103.0蒜氨酸提取完全需要一定時(shí)間，故考察使提取率最高的最優(yōu)提取時(shí)提取時(shí)間（min）樣品編號(hào)稱樣量峰面積蒜氨酸(%)平均含量RSD(%)提取率312311.02181.02761.01481.04172695762682822665182773230.4130.4080.4110.4150.4110.52102.752311.05031.08131.03142823732867442685130.4190.4130.4070.4160.71104.0231.04801.02432750602706240.4110.4130.4100.74102.620mL，提取5min，置50mL容量瓶中，加預(yù)熱水淋洗容器，洗液合并置容量瓶中，室溫冷卻，加水稀釋至刻度，搖勻，濾紙過(guò)濾。取續(xù)濾液2.大蒜及其制品方法學(xué)考察蒜氨酸μg/mLAS/N22.7336270734.7411.36517923018.829.09214661015.666.36410095810.36定量限4.546728837.312.273357983.661.137175601.818287393.07檢測(cè)限6.1379803510.996.81910892511.182.046323253.261.591247522.65配制系列蒜氨酸標(biāo)準(zhǔn)溶液的濃度為2.273、4.546、9.092、22.73、蒜氨酸濃度μg/mL峰面積AA1A2A3A2.273352843508334927350984.546716427168071837717209.09214423614441314446714437222.7335953736000235990935981645.4672232772265872293872264190.921445423144423814448511444837由圖可知，得回歸方程A=15901C-746.34，r=1，結(jié)果表明，蒜氨酸編號(hào)取樣量(g)A(蒜氨酸)含量(%)含量(%)RSD(%)150.2096486876250.2990481724350.31094992824.3450.0997482627550.2780538927650.1038507175編號(hào)取樣量(g)Aalliin含量(%)平均含量(%)RSD(%)10.30233985874.1420.30284021324.1730.30604043764.154.150.7540.30464006124.1350.30393976534.1160.30344055354.20編號(hào)稱樣量(g)A(蒜氨酸)含量(%)平均含量(%)RSD(%)11.01442668240.41321.03432678080.4070.40931.01632660800.41141.01762605040.40251.01912698760.41661.03812695660.408人員編號(hào)取樣量(g)A(蒜氨酸)含量(%)含量(%)RSD(%)含量(%)RSD%A人員12345650.209650.299050.310950.099750.278050.10384868764817244992824826275389275071754.34.312350.858050.311450.7350518820504251518686B人員45650.858050.311450.73505012875650555265704.7人員編號(hào)取樣量(g)A(蒜氨酸)含量(%)含量(%)RSD(%)含量(%)RSD%A人員1234560.30230.30280.30600.30460.30390.30343985874021324043764006123976534055354.144.174.154.134.114.204.150.754.111230.30640.30490.30594127774051534137394.064.004.07B人員4560.30460.30200.30654163704110484189584.124.104.124.08人員編號(hào)取樣量(g)A(蒜氨酸)含量(%)含量(%)RSD(%)含量(%)RSD%A人員11.01442668240.4130.4091.20.4111.021.03432678080.40734561.01631.01761.01911.03812660802605042698762695660.4110.4020.4160.40811.02372784840.41021.02022823890.41731.02412794990.411B人員0.412B人員41.02932784720.40851.01442753350.40961.01202790100.415量瓶中，分別加入蒜氨酸對(duì)照品溶液（0.958mg/mL）0mL、0.8mLmL、1.2mL，加水稀釋至刻度，搖勻，即得。按外標(biāo)法以峰面積編號(hào)取樣量本底值加入量實(shí)測(cè)值平均回RSD50.9891//469.61/本底50.9093//464.59///50.7793//443.92/50.7257458.05824.3998.0本底+低50.9270459.87383.2846.23100.4100.01.850.9787460.34856.32101.5本底+中50.7632458.39916.4097.8100.13.3本底+高50.978850.718050.942550.873050.6225460.34457.98460.01459.38457.12479.0574.8975.19924.841074.51071.61068.9103.898.7103.8103.6103.6103.70.13低、中、高濃度蒜氨酸回收率分別為100.0%加無(wú)水甲醇20mL，混勻，分別加入濃度為4.28mg/mL的蒜氨酸對(duì)照品編號(hào)取樣量本底值加入量實(shí)測(cè)值平均回RSD本底0.30620.30680.30190.3011///12.59///12.6012.8712.7922.36///100.1//本底+低0.30230.30530.306312.6412.7612.809.7422.4422.4424.38100.399.799.6100.10.29本底+中0.30580.30650.307112.7812.8112.8411.6924.0524.4126.5098.399.6100.199.10.77本底+高0.30550.306212.7712.8013.6426.4526.39100.299.8100.00.18取本品12份，每份約1.0g，精密稱量瓶中，用55℃的水淋洗容器，洗液合并置量瓶中，放冷，加水稀釋至編號(hào)取樣量本底值加入量實(shí)測(cè)值平均回RSD本底1.03721.03031.08801.0384///4.32///4.334.264.537.85///101.6//本底+低1.03691.05681.06314.314.404.423.428.098.108.70104.8103.9100.2103.4本底+中1.08231.05391.07614.504.384.484.288.818.669.51100.5100.399.1100.30.19本底+高1.06021.04084.414.335.149.669.36101.499.199.9考察在測(cè)定條件下試驗(yàn)參數(shù)發(fā)生細(xì)小改變時(shí),檢測(cè)不受影響的能力。改變條件RT含量（%）平均含量（%）RSD(%）柱溫20℃12.941柱溫25℃12.5550.55柱溫30℃12.158甲醇：0.06%TFA（0:100）12.555甲醇：0.06%TF（2.5:97.5）12.511甲醇：0.06%TFA（5:95）9.3090.4mL/min15.6270.5mL/min12.5550.6mL/min10.467柱子型號(hào)：GIST12.555柱子型號(hào)：島津AQ-C1813.1450.64柱子型號(hào)：依利特ODS-C189.908改變條件RT含量（%）平均含量（%）RSD(%）柱溫20℃12.9203.753.730.46柱溫25℃12.5353.72114030114030柱溫30℃12.1383.72甲醇：0.06%TFA（0:100）12.5353.72甲醇：0.06%TF（2.5:97.5）甲醇：0.06%TFA（5:95）12.5229.2913.843.363.646.90.4mL/min15.6023.790.5mL/min12.5353.723.770.6mL/min10.4513.80柱子型號(hào)：GIST12.5353.72柱子型號(hào)：島津AQ-C1813.1393.593.622.3柱子型號(hào)：依利特ODS-C189.9123.56色譜柱型號(hào)為：Shim-packGIST改變條件RT含量（%）平均含量（%）RSD(%）柱溫20℃12.9220.412柱溫25℃12.5360.4080.4110.64柱溫30℃12.1390.413甲醇：0.06%TFA（0:100）12.5360.408甲醇：0.06%TF（2.5:97.5）12.5030.4220.4064.2甲醇：0.06%TFA（5:95）9.2950.3880.4mL/min15.6040.4160.5mL/min12.5360.4080.4140.6mL/min10.4500.419柱子型號(hào)：GIST12.5360.408柱子型號(hào)：島津AQ-C1813.1400.403..柱子型號(hào)：依利特ODS-C189.9080.399放置時(shí)間峰面積蒜氨酸含量%降解率%RSD(%)0526694/25240310.5145237490.5685229450.715222380.85520939245218860.91結(jié)果顯示，不同時(shí)間點(diǎn)測(cè)得的樣品中蒜氨酸降解率小于2.0%，表明2.7.2大蒜粉樣品溶液的穩(wěn)定性放置時(shí)間峰面積蒜氨酸含量%降解率%RSD(%)02483768853.89/3771103.90-0.060.373768373.890.013763563.890.143755393.880.363742413.870.70243736643.860.85結(jié)果顯示，不同時(shí)間點(diǎn)測(cè)得的樣品中蒜氨酸降解率小于1.0%，表明放置時(shí)間峰面積蒜氨酸含量%降解率%RSD(%)02794890.426/22701590.4123.3442705090.4133.2182686610.4103.872699690.4123.412694570.4113.59242697900.4123.47結(jié)果顯示，不同時(shí)間點(diǎn)測(cè)得的樣品中蒜氨酸降解率小于2.0%，表明2.8.1大蒜樣品方法驗(yàn)證編號(hào)取樣量(g)A含量(%)平均含量(%)RSD(%)1250.763450.245650.259050.02064239334045704141263371654.550.963650.134650.52893175963132846829280.980.992.13350.958950.993050.45866391166375164056622.034.5450.493350.846250.36983853013808733405083.7550.458150.173250.26973215093125984282360.984.3650.255950.020650.36364036384171532998350.943.0750.134650.178950.15892806502915583855500.880.920.9143.1850.393050.455650.43333648463723262578690.813.1950.526250.369250.42812449642500794313810.770.780.7862.750.175550.264750.25494020604165495119943.350.257850.26794838405044412.9編號(hào)取樣量(g)A含量(%)平均含量(%)RSD(%)1234567890.30730.30670.30440.30740.30650.30310.30900.30910.30950.30790.30570.30680.30140.30410.30260.30340.30430.30580.30620.30410.30670.30800.30820.30880.30230.30210.30350.30650.30663812753743283692093788363756743699333759523715703729013322053257133273692704772663432671502821382819432814124038053977464049232371112350422341482707262660352706602883682905783.883.823.793.853.833.823.813.763.773.373.333.342.812.742.762.912.902.884.124.094.132.412.392.372.802.752.792.942.963.833.843.783.352.772.904.122.392.782.960.490.640.690.530.510.770.870.480.30680.30340.30510.30212912424093634200734135042.974.224.314.284.27編號(hào)取樣量(g)A含量(%)平均含量(%)RSD(%)12345671.00441.01261.02691.08791.06851.04081.05041.04361.06111.02131.00031.03311.05531.05601.03691.08581.08231.06251.00821.03661842481846741878092004761972041927423043563042913144092618162482672627571972221940121951352598672621312597143074243127370.2890.2870.2880.2900.2910.2920.4560.4590.4670.4040.3910.4010.2940.2890.2960.3770.3820.3850.4800.4750.2880.2910.4610.3990.2930.3810.4780.290.250.54精密精密度1.04801.07103184802605440.4790.38381.00171.06551.06012499742607442616770.3930.3860.3890.38791.03241.02561.04092644302599831962190.4040.3990.2970.3971.04031.02851.02641983431960252558920.3000.3000.3930.2990.641.03941.02972634152601460.3990.3980.3970.8711批大蒜軟膏劑樣品測(cè)定結(jié)果準(zhǔn)確度高，重復(fù)性好，蒜氨酸含量的驗(yàn)證結(jié)果專屬性空白試驗(yàn)系統(tǒng)適用性試驗(yàn)該色譜條件下陰性對(duì)照溶液對(duì)蒜氨酸峰無(wú)影響，供試品分離良好。以蒜氨酸峰計(jì)算理論塔板數(shù)為21750，蒜氨酸峰與相鄰峰的分離度為12.089，達(dá)到基線分離，符合相關(guān)規(guī)定。蒜氨酸峰的拖尾因子為1.110，符合相關(guān)規(guī)定。線性和范圍以蒜氨酸對(duì)照品濃度C為橫坐標(biāo)，峰面積A為縱坐標(biāo)線性回歸，得回歸方程A=15901C-746.34，相關(guān)系數(shù)r=1.000，結(jié)果表明，蒜氨酸在2.27-90.92μg/mL濃度范圍內(nèi)線性關(guān)系良好。準(zhǔn)確度大蒜中蒜氨酸含量為0.903%，《中國(guó)藥典》規(guī)定回收率限度為92%-105%。測(cè)定高中低3個(gè)濃度，回收率均在92.0-105.0%之間，平均回收率分別為101.3%，RSD（n=9）為0.07%。重復(fù)精密度中間精密度測(cè)定6份供試品溶液中蒜氨酸含量的測(cè)定結(jié)果基本一致，蒜氨酸百分含量的RSD為4.3%，表明該方法重復(fù)性良好。由兩名分析人員，分別測(cè)定同一份供試品，A人員RSD（n=6）為4.3%；B人員RSD（n=6）為4.7%。符合藥典規(guī)定。溶液的穩(wěn)定性供試品溶液放置0、2、4、6、8、12和24h，進(jìn)樣主成分峰面積無(wú)明顯變化，RSD（n=7）為1.4%，結(jié)果顯示含量測(cè)定溶液至少在24h內(nèi)保持穩(wěn)定。耐用性將色譜條件中的參數(shù)作適度變化后，如色譜條件中的流速、柱溫參數(shù)作微小調(diào)整對(duì)測(cè)定結(jié)果基本不會(huì)產(chǎn)生顯著性影響，但改變色譜柱的廠家及流動(dòng)相的比例對(duì)蒜氨酸的測(cè)定結(jié)果有一定的影響，故需要特殊說(shuō)明，并寫(xiě)入標(biāo)準(zhǔn)中。方法驗(yàn)證11批大蒜樣品測(cè)定結(jié)果準(zhǔn)確度高，重復(fù)性好，蒜氨酸含量的RSD%均符合藥典要求。驗(yàn)證結(jié)果專屬性空白試驗(yàn)系統(tǒng)適用性試驗(yàn)該色譜條件下陰性對(duì)照溶液對(duì)蒜氨酸峰無(wú)影響，供試品分離良好。以蒜氨酸峰計(jì)算理論塔板數(shù)為21750，蒜氨酸峰與相鄰峰的分離度為12.089，達(dá)到基線分離，符合相關(guān)規(guī)定。蒜氨酸峰的拖尾因子為1.110，符合相關(guān)規(guī)定。線性和范圍以蒜氨酸對(duì)照品濃度C為橫坐標(biāo)，峰面積A為縱坐標(biāo)線性回歸，得回歸方程A=15901C-746.34，相關(guān)系數(shù)r=1.000，結(jié)果表明，蒜氨酸在2.27-90.92μg/mL濃度范圍