超高效液相色谱-串联质谱法测定阿胶中的驴皮源成分

近年来由于驴皮资源短缺,阿胶价格大幅度上涨,市场上出现了大量以马、骡、猪、牛皮等熬制而成的假胶,导致阿胶质量的参差不齐,严重扰乱了市场,急需高效准确的检测方法提升阿胶品质。该研究采用超高效液相色谱-串联质谱技术,建立了阿胶中驴皮源成分的检测方法。样品加水溶解后,于37 ℃下经胰蛋白酶酶解,产生驴源性特征肽段,以0.1%甲酸乙腈溶液和0.1%甲酸水溶液作为流动相进行梯度洗脱,单次分析时间10 min,在电喷雾正离子(ESI⁺)模式下进行多反应监测(MRM),同位素内标法定量。驴源多肽A1、A2在50~1250 μg/L范围内线性关系良好,相关系数(r)均大于0.996,方法定量限(S/N=10)为20 mg/kg,在300、600、900 mg/kg 3个添加水平上驴源多肽A1、A2的回收率范围为103.2%~108.3%,各加标水平平行测定结果的相对标准偏差(RSD)为1.0%~3.0%,完全能够满足实际样品检测需求。对29批不同生产企业的阿胶进行测定,结果表明,不同企业生产的阿胶中驴源多肽A1、A2的含量之和存在差异,含量为0.096%~0.180%,平均值为0.151%,提示驴源多肽A1、A2含量较低的阿胶生产厂家应注重皮源质量,改进生产工艺,以提升产品质量。该方法操作简便,结果可靠,重现性好,可用于阿胶中驴皮源成分的测定。     

在线二维液相色谱-四极杆飞行时间质谱法检测头孢噻吩钠的杂质谱

建立了在线二维液相色谱-四极杆飞行时间质谱检测头孢噻吩钠杂质谱的方法,有效地解决了流动相中含不挥发性磷酸盐的色谱系统不适合用于液相色谱-质谱快速鉴定杂质的难题。一维高效液相色谱(HPLC)以Symmetry C18为色谱柱,以磷酸盐缓冲液(pH 2.5)和乙腈梯度洗脱;二维以ACQUITY UPLC BEH C18为色谱柱,以0.1%(v/v)甲酸水溶液和0.1%(v/v)甲酸乙腈溶液梯度洗脱。以HLB C18为捕集柱,用0.1%(v/v)甲酸水溶液进行捕集和脱盐,采用正离子模式采集数据。对头孢噻吩钠中6个杂质进行了结构鉴定,对其来源进行了分析,并进一步确证了《中国药典》2010年版对头孢噻吩钠杂质A认定有误。采用本方法可以快速、简便、灵敏地对头孢噻吩钠杂质谱进行检测。     

高效液相色谱-串联质谱法同时测定贝类中22种农药残留

建立了高效液相色谱-三重四极杆质谱（HPLC-MS/MS）同时测定贝类中22种农药残留的分析方法。样品经含0.1%（v/v）甲酸的乙腈提取，N-丙基乙二胺（PSA）和石墨化碳黑（GCB）净化，然后采用ACE UltraCore 2.5 SuperC18柱（100 mm×2.1 mm，2.5 μm）分离，以甲醇-0.1%（v/v）甲酸水溶液为流动相梯度洗脱，流速为0.4 mL/min，柱温为35 ℃，然后以电喷雾电离（ESI）源，在多反应监测（MRM）、正离子模式下，采用三重四极杆质谱检测。22种农药在各自的线性范围内线性关系良好，相关系数均大于0.997，检出限为0.1~0.3 μg/kg，定量限为0.3~1.0 μg/kg。在3个添加水平下，22种农药的平均回收率为65.2%~109.4%，相对标准偏差为1.3%~15.2%（n=6）。该方法操作简单，快速，准确度高，灵敏度高，可用于贝类中22种农药残留的同时检测。     

超高效液相色谱-串联质谱法测定人血浆中盐酸氨溴索及其制剂的生物等效性评价

采用超高效液相色谱-串联质谱（UHPLC-MS/MS）技术,建立了快速、简单、灵敏的测定人血浆中盐酸氨溴索含量的方法,并用于盐酸氨溴索人体生物等效性预试验研究。取50 μL血浆样品,采用蛋白沉淀法处理,以盐酸氨溴索-d5为内标。采用Waters XBridge BEH C18色谱柱（50 mm×2.1 mm,2.5 μm）,以0.1%（v/v）甲酸水-含0.1%（v/v）甲酸的甲醇为流动相,在0.4 mL/min流速下进行梯度洗脱。采用电喷雾电离（ESI）源以正离子模式进行MRM检测。结果显示,盐酸氨溴索在2~400 ng/mL范围内线性关系良好,相关系数（r）为0.998,准确度为97.1%~108.7%,精密度为1.0%~5.6%。将该方法用于6名健康受试者口服盐酸氨溴索受试制剂和参比制剂30 mg后血药浓度的测定,结果显示二者相对生物利用度为（102.3±14.8）%,血药浓度-时间曲线下面积（AUC_0-t、AUC_0-∞）和最大血药浓度（C_max）的90%置信区间均在80.0%~125.0%范围内,两种制剂生物等效。     

固相萃取-超快速液相色谱-串联质谱法同时测定血液中的4种苏丹染料

建立了血液样品中4种苏丹染料（苏丹Ⅰ、Ⅱ、Ⅲ和Ⅳ）的固相萃取-超快速液相色谱-串联质谱（UFLC-MS/MS）测定方法。样品经乙腈涡旋振荡提取,上清液加等体积水稀释混匀后移入C18固相萃取小柱净化,采用Agilent Eclipse Plus C18色谱柱（100 mm×2.1 mm,1.8 μm）以0.1%（v/v）甲酸水溶液和0.1%（v/v）甲酸乙腈溶液为流动相梯度洗脱分离,电喷雾电离（ESI）正离子多反应监测模式进行定量分析。讨论了苏丹Ⅲ和苏丹Ⅳ的偶氮基团E-Z光学异构现象,并对影响因素进行了分析。结果4种苏丹染料在0.1~20.0 μg/L范围内线性关系良好,相关系数均大于0.999;在低、中、高3个加标水平的平均回收率为93.0%~108.2%,相对标准偏差为4.8%~9.5%;方法的检出限（LOD）为0.06 μg/L,定量限（LOQ）为0.2 μg/L。本方法准确、快速、灵敏,可用于血液样品中苏丹类染料的检测分析。     

液相色谱-串联质谱法测定食用植物油中的棉酚

建立了食用植物油中棉酚的液相色谱-串联质谱（LC-MS/MS）分析方法。待测物经无水乙醇涡旋振荡提取,C18色谱柱分离,以乙腈和0.1%（v/v）甲酸水溶液为流动相进行梯度洗脱,LC-MS/MS测定,外标法定量。方法的测定低限（S/N>10）为1 mg/kg;在添加浓度为1、2和200 mg/kg水平下,棉酚的加标回收率为87.4%~100%,相对标准偏差为3.9%~12.2%。结果表明,本方法灵敏度高,测定结果准确,回收率稳定,可用于食用植物油中棉酚残留的确证检测。     

超高效液相色谱-串联质谱法同时测定保健食品中丙磺舒、别嘌醇和苯溴马隆

建立了基于QuEChERS前处理的超高效液相色谱-串联质谱（UPLC-MS/MS）同时测定抗痛风类保健食品中别嘌醇、丙磺舒和苯溴马隆3种药物的检测方法。样品经含0.1%（v/v）氨水的乙腈溶液超声提取后，采用乙二胺-N-丙基硅烷（PSA）和十八烷基键合硅胶（C₁₈）吸附剂净化，用C₁₈色谱柱进行分离，0.1%（v/v）甲酸水溶液和甲醇为流动相进行梯度洗脱，采用电喷雾电离源、正负离子切换模式和多反应监测（MRM）模式检测。结果表明，别嘌醇、丙磺舒和苯溴马隆的检出限分别为5、25和25 μg/kg，定量限分别为17、80和80 μg/kg。抗痛风类保健食品中3种药物的平均加标回收率为76.8%~116.6%，相对标准偏差（RSD）为2.7%~14.6%。应用该法对68批次保健食品进行分析，其中1批次样品检出别嘌醇药物。该法操作简单，灵敏度高，可用于抗痛风类保健食品中别嘌醇、丙磺舒和苯溴马隆的测定。     

高效液相色谱-四极杆飞行时间质谱法同时检测豆芽中的3种外源植物激素残留

建立了高效液相色谱-四极杆飞行时间质谱同时测定豆芽中6-苄基腺嘌呤、4-氯苯氧乙酸、赤霉素等3种外源植物激素残留的方法。采用QuEChERS前处理技术,豆芽样品以酸化乙醇-乙腈溶液（1%（v/v,下同）乙酸+50%乙醇+49%乙腈）提取目标化合物,并经乙腈再提取后合并提取液;经硅藻土分散固相净化、正己烷去脂后氮吹至近干;用2.0 mL 50%（v/v）甲醇水溶液定容,过滤膜后上机检测。液相色谱以甲醇-水（含0.1%（v/v）的甲酸）作为流动相梯度洗脱,C₁₈色谱柱分离;质谱采用高分辨质谱、负离子模式,以精确质量数和二级特征离子定性,以准分子离子峰面积定量。结果表明3种目标化合物的定量限为5.0~10.0 μg/kg,线性范围为1~200 μg/L;平均添加回收率为79.1%~96.1%,相对标准偏差为5.7%~10.4%。本方法具有操作简单、快捷、灵敏度高等优点,适用于市场上豆芽质量的快速筛查检测。     

QuEChERS-超高效液相色谱-四极杆/静电场轨道阱高分辨质谱快速测定水产品中25种药物残留

采用了一种高效基质脂肪吸附剂（EMR-Lipid）去除水产品基质中的脂肪和磷脂等杂质,利用超高效液相色谱-四极杆/静电场轨道阱高分辨质谱法（UPLC-Q Orbitrap HRMS）的同时定性定量功能,建立了水产品中25种药物残留的检测方法。样品经乙腈提取,EMR-Lipid净化,同时加入3 g氯化钠和3 g无水硫酸钠进行盐析,采用ACQUITY UPLC BEH C18色谱柱（100 mm×2.1 mm,1.7 μm）分离,以含0.1%（v/v）甲酸的乙腈溶液和0.1%（v/v）甲酸水溶液为流动相进行梯度洗脱,在加热电喷雾离子（HESI）源、全扫描/实时二级质谱扫描（Full MS/dd-MS²）监测模式下进行检测。结果表明,25种目标化合物的质量浓度与母离子峰面积间的线性关系良好,相关系数（r）均大于0.997;25种目标化合物的检出限为0.1~1.0 μg/kg,其平均加标回收率为70.1%~108.9%,相对标准偏差为2.1%~13.8%。该法具有操作简单快捷、灵敏度高等优点。     

超高效液相色谱-串联质谱同时测定植物油中14种营养成分

建立超高效液相色谱-串联质谱同时检测植物油中4种生育酚、4种生育三烯酚、4种植物甾醇、β-胡萝卜素和角鲨烯等14种营养成分的方法。样品经皂化处理后,采用石油醚提取浓缩,用甲醇定容。采用Poroshell 120 PFP色谱柱（150 mm×3.0 mm,2.7 μm）分离,以0.1%（v/v）甲酸水溶液和0.1%（v/v）甲酸甲醇溶液为流动相进行梯度洗脱,流速为0.3 mL/min。采用大气压化学电离源、正离子模式,在选择反应监测模式下扫描。结果表明,14种营养成分在0.05~10.0 mg/L范围内相关系数≥0.9971;在不同添加水平下,14种营养成分的回收率为80.7%~100.5%,相对标准偏差＜6.0%（n=6）;方法的检出限和定量限分别为0.01~0.30 μg/g和0.04~1.00 μg/g。该法灵敏、准确,分析时间快,稳定性好,适用于植物油中14种营养成分的同时检测。     

Qualitative and Quantitative Analysis of Ejiao-Related Animal Gelatins through Peptide Markers Using LC-QTOF-MS/MS and Scheduled Multiple Reaction Monitoring (MRM) by LC-QQQ-MS/MS

Donkey-hide gelatin, also called Ejiao (colla corii asini), is commonly used as a food health supplement and valuable Chinese medicine. Its growing popular demand and short supply make it a target for fraud, and many other animal gelatins can be found as adulterants. Authentication remains a quality concern. Peptide markers were developed by searching the protein database. However, donkeys and horses share the same database, and there is no specific marker for donkeys. Here, solutions are sought following a database-independent strategy. The peptide profiles of authentic samples of different animal gelatins were compared using LC-QTOF-MS/MS. Fourteen specific markers, including four donkey-specific, one horse-specific, three cattle-specific, and six pig-specific peptides, were successfully found. As these donkey-specific peptides are not included in the current proteomics database, their sequences were determined by de novo sequencing. A quantitative LC-QQQ multiple reaction monitoring (MRM) method was further developed to achieve highly sensitive and selective analysis. The specificity and applicability of these markers were confirmed by testing multiple authentic samples and 110 batches of commercial Ejiao products, 57 of which were found to be unqualified. These results suggest that these markers are specific and accurate for authentication purposes.     

Species-specific polymerase chain reaction amplification of camel (Camelus) DNA extracts

A sensitive polymerase chain reaction (PCR) method based on amplification of a specific DNA fragment was established for the identification of camel (Camelus) materials. The species-specific primer pair L183/H372 was designed based on the nucleotide sequence of the mitochondrial cytochrome b gene, and its specificity was confirmed by amplification of 3 camel (domestic double-humped camel, wild double-humped camel, wild one-humped camel) samples and 11 non-Camelus animal (sheep, goat, pig, chicken, cattle, fish, dog, horse, donkey, deer, and rabbit) materials. An expected 208 base pair fragment was amplified from camel materials; no cross-reactive or additional fragments were generated from other animal materials. Taq I restriction endonuclease digestion of the unpurified PCR product can be used routinely to confirm the camel origin of the amplified sequence.     

Gas-phase separations of complex tryptic peptide mixtures

High-resolution ion mobility and time-of-flight mass spectrometry techniques have been used to analyze complex mixtures of peptides generated from tryptic digestion of fourteen common proteins (albumin, bovine, dog, horse, pig, and sheep; aldolase, rabbit; beta-casein, bovine; cytochrome c, horse; beta-lactoglobulin, bovine; myoglobin, horse; hemoglobin, human, pig, rabbit, and sheep). In this approach, ions are separated based on differences in mobilities in helium in a drift tube and on differences in their mass-to-charge ratios in a mass spectrometer. From data recorded for fourteen individual proteins (over a m/z range of 405 to 1,000), we observe 428 peaks, of which 205 are assigned to fragments that are expected from tryptic digestion. In a separate analysis, the fourteen mixtures have been combined and analyzed as one system. In the single dataset, we resolve 260 features and are able to assign 168 peaks to unique peptide sequences. Many other unresolved features are observed. Methods for assigning peptides based on the use of m/z information and existing mobilities or mobilities that are predicted by use of intrinsic size parameters are described.     

分散固相萃取-超高效液相色谱-串联质谱法测定禽蛋中氟虫腈及其代谢产物

建立了分散固相萃取-超高效液相色谱-串联质谱检测禽蛋中氟虫腈及其代谢产物氟甲腈、氟虫腈硫醚和氟虫腈砜的方法。样品用含1%(体积分数)乙酸的乙腈提取后,加无水硫酸钠、十八烷基键合硅胶(C18-N)及氨基-丙基乙二胺(NH2-PSA)3种净化剂,旋涡振荡,对样品进行净化。以Shim-pack GIST C18色谱柱(50 mm×2.1 mm,2μm)进行分离,以甲醇和1 mmol/L乙酸铵水溶液为流动相进行梯度洗脱,在电喷雾离子源负离子模式和多反应检测(MRM)模式下进行定性定量分析。考察了净化剂中无水硫酸钠、C18-N和NH2-PSA的用量对加标回收率的影响,优化了实验条件。结果表明:4种化合物在0.4~100μg/L范围内线性关系良好,相关系数r2≥0.998 9;回收率为95.09%~103.26%;定量限为0.2μg/kg。该方法前处理简单,回收率高,重复性好,可作为禽蛋中氟虫腈及其代谢产物的有效检测方法。     

Rapid multi-residue screening of antibiotics in muscle and kidney by liquid chromatography-electrospray ionization-tandem mass spectrometry

A new approach for simple and rapid multi-residue screening of antibiotics in muscle and kidney by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is described. Nineteen analytes from five classes of antibiotics, i.e. tetracyclines, sulfonamides, quinolones, beta-lactams and macrolides were included in the method. The recently registered drug acetylisovaleryltylosin and its metabolite 3-ortho-acetyltylosin, for which no methods of analysis have been published so far, are among the analytes. The samples were extracted by a single extraction using 70% methanol, diluted with water and injected in the LC-MS/MS. The total run time for each sample was 15 min. At least 60 samples could be analysed and evaluated in 24 h. The accuracy of the method is sufficiently good for screening of antibiotics at the maximum residue limit (MRL) in both muscle and kidney from pig, cattle, sheep, deer, horse and reindeer. Validation was performed according to Commission Decision 2002/657/EC. In addition, a possible way of rapid and simple confirmation of the analytes in muscle is suggested.     

超高效液相色谱-串联质谱法同时检测口腔卫生用品中的硝基咪唑类药物

建立了采用超高效液相色谱-串联质谱同时检测口腔卫生用品(牙膏及漱口水)中甲硝唑、替硝唑、奥硝唑、二甲硝咪唑和洛硝唑的方法。试样以0.1%(体积分数)的甲酸水溶液/乙腈(95:5, v/v)稀释,经高速离心后过滤膜净化,采用Cloversil C18色谱柱(100 mm×2.1 mm, 3.5 μm)分离,以0.1%甲酸水溶液和乙腈为流动相梯度洗脱,质谱检测,外标法定量。5种硝基咪唑类化合物在1.0～60.0 μg/L质量浓度范围内线性关系良好,相关系数r均不小于0.9992;在10.0、20.0和100 mg/kg加标水平的平均回收率为91.5%～108%,相对标准偏差为1.14%～5.22%;方法的定量限(LOQ,以信噪比为10计)为2.0 mg/kg。该方法可靠、稳定,可满足口腔卫生用品中硝基咪唑类药物含量检测与确证的需要。     

高效液相色谱-串联质谱法测定动物源性食品中矮壮素残留

建立了高效液相色谱-串联质谱测定动物源性食品中矮壮素残留的分析方法。样品经含1%（v/v）乙酸的乙腈溶液提取、正己烷脱脂、阳离子固相萃取柱（PCX）净化,采用Venusil MP C18（2）色谱柱（150 mm×2.1 mm,3 μm）分离,以乙腈和0.1%（v/v）甲酸水溶液为流动相进行梯度洗脱,采用电喷雾电离、正离子模式扫描,多反应监测模式（MRM）检测,基质匹配标准曲线内标法定量。结果表明：矮壮素在0.200~500 μg/L范围内呈良好线性,相关系数（r²）均不低于0.9993,方法的定量限为0.500 μg/kg;以猪肉、牛肉、羊肉、鸡肉、鸡蛋、猪肾、牛肝、羊肾、鸡肝、牛奶为基质,矮壮素的平均加标回收率为93.4%~101%,相对标准偏差为2.3%~8.0%。该方法基质干扰小,灵敏度高,准确可靠,适用于动物源性食品中矮壮素残留的定量检测。     

Gas-phase separations of complex tryptic peptide mixtures

High-resolution ion mobility and time-of-flight mass spectrometry techniques have been used to analyze complex mixtures of peptides generated from tryptic digestion of fourteen common proteins (albumin, bovine, dog, horse, pig, and sheep; aldolase, rabbit; β-casein, bovine; cytochrome c, horse; β–lactoglobulin, bovine; myoglobin, horse; hemoglobin, human, pig, rabbit, and sheep). In this approach, ions are separated based on differences in mobilities in helium in a drift tube and on differences in their mass-to-charge ratios in a mass spectrometer. From data recorded for fourteen individual proteins (over a m/z range of 405 to 1000), we observe 428 peaks, of which 205 are assigned to fragments that are expected from tryptic digestion. In a separate analysis, the fourteen mixtures have been combined and analyzed as one system. In the single dataset, we resolve 260 features and are able to assign 168 peaks to unique peptide sequences. Many other unresolved features are observed. Methods for assigning peptides based on the use of m/z information and existing mobilities or mobilities that are predicted by use of intrinsic size parameters are described. Received: 26 August 2000 / Revised: 15 November 2000 / Accepted: 16 November 2000     

高效液相色谱-串联质谱法同时测定血液中的15种减肥药物

建立了一种专属、灵敏的同时测定血液中咖啡因、盐酸西布曲明等15种减肥药的高效液相色谱-串联质谱(HPLC-MS/MS)分析方法。样品经乙腈沉淀后,进入HPLC-MS/MS中分析检测。以甲醇和含0.1%(v/v)冰醋酸的20 mmol/L醋酸铵溶液作为流动相,采用梯度洗脱方式,以UltimateXB-C18为色谱柱进行HPLC分析;质谱分析采用电喷雾离子源,正负离子快速切换扫描,选择反应监测模式检测。15种减肥药的定量限在0.001～0.05 mg/L内,各种药物的灵敏度较高,各成分的线性相关系数均大于0.99,精密度均小于12.3%,回收率范围为77.3%～110.8%。研究了这15种药物的质谱特征。该方法灵敏、简便、快捷、专属性强,可用于动物实验样品中减肥药物的含量测定,并且对其他药品、食品中目标减肥药物的测定具有借鉴意义。