超高效液相色谱-串联质谱法测定辅助生殖用培养液中谷氨酰胺的含量
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摘要
背景:应用高效液相色谱-串联质谱法检测生物样本中氨基酸含量具有样品处理简单、分析时间短、高选择性、高灵敏度等优势,但目前该方法主要用于脑透析液、尿液及血清中谷氨酰胺含量的测试。目的:采用超高效液相色谱-串联质谱法测定辅助生殖用培养液中的谷氨酰胺含量。方法:采用超高效液相色谱-串联质谱仪,色谱柱为SUPELCO Discovery HS F5-3(15 cm×2.1 mm,3μm),柱温40℃,以0.1%甲酸水溶液-0.1%甲酸乙腈溶液为流动相,梯度洗脱,流速0.35mL/min;电喷雾离子化源负离子模式扫描,采用多反应监测模式检测谷氨酰胺含量。结果与结论:谷氨酰胺在0.123 7-24.74 mg/L范围内线性关系良好(r=0.999 7),平均回收率为102.9%-108.2%,相对标准偏差RSD为2.3%-4.9%,谷氨酰胺定量限为9.76μg/L。将受精培养液(含谷氨酰胺)在37℃下放置24,96h的谷氨酰胺含量较原液分别下降了6%和15%。结果说明采用高效液相色谱-串联质谱法检测辅助生殖用培养液中的谷氨酰胺含量,操作简单,不需要对样品进行衍生化处理,方法专属性、准确性及灵敏度良好,且测试时间短,可用于辅助生殖用培养液的质量控制,研究添加成分谷氨酰胺的降解而产生的临床风险。
BACKGROUND: Ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for analyzing amino acids in biological samples is simple in sample preparation with a short analysis time, and has high sensitivity and specificity. Until now, it is only applied in analyzing glutamine contents in dialysate, urine and plasma. OBJECTIVE: To establish a method for determining glutamine concentration in human assisted reproductive media by UPLC-MS/MS. METHODS: The UPLC separation was performed on a SUPELCO Discovery HS F5-3 column (15 cm×2.1 mm, 3 μm) at 40 oC. The mobile phase was acetonitrile and water (both containing 0.1% formic acid) in a gradient elute mode. The flow rate was 0.35 mL/min. Electrospray ionization with a negative-ion and multiple reaction monitoring mode was used. RESULTS AND CONCLUSION: The linearity was achieved in the range of 0.123 7-24.74 mg/L for glutamine (r=0.999 7). The recoveries were 102.9%-108.2% with the range 2.3%-4.9% for the relative standard deviation. The limit of qualification was 9.76 μ g/L. The fertilization culture medium containing glutamine was incubated at 37 oC for 96 hours, in which the glutamine content declined 6% at 24 hours and 15% at 96 hours respectively compared with initial content. Therefore, the method is simple, specific, accurate and sensitive without sample derivation, and the test time is short. It is suitable for the quality control of human assisted reproductive media and useful for the risk study related to the degradation of glutamine.
BACKGROUND: Ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for analyzing amino acids in biological samples is simple in sample preparation with a short analysis time, and has high sensitivity and specificity. Until now, it is only applied in analyzing glutamine contents in dialysate, urine and plasma. OBJECTIVE: To establish a method for determining glutamine concentration in human assisted reproductive media by UPLC-MS/MS. METHODS: The UPLC separation was performed on a SUPELCO Discovery HS F5-3 column (15 cm×2.1 mm, 3 μm) at 40 oC. The mobile phase was acetonitrile and water (both containing 0.1% formic acid) in a gradient elute mode. The flow rate was 0.35 mL/min. Electrospray ionization with a negative-ion and multiple reaction monitoring mode was used. RESULTS AND CONCLUSION: The linearity was achieved in the range of 0.123 7-24.74 mg/L for glutamine (r=0.999 7). The recoveries were 102.9%-108.2% with the range 2.3%-4.9% for the relative standard deviation. The limit of qualification was 9.76 μ g/L. The fertilization culture medium containing glutamine was incubated at 37 oC for 96 hours, in which the glutamine content declined 6% at 24 hours and 15% at 96 hours respectively compared with initial content. Therefore, the method is simple, specific, accurate and sensitive without sample derivation, and the test time is short. It is suitable for the quality control of human assisted reproductive media and useful for the risk study related to the degradation of glutamine.
引文
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[15]刘婷婷,邢青斌,程佳丽,等.桦褐孔菌氨基酸含量测定方法的建立[J].营养学报,2017,39(5):504-506.
[16]陈思,郜玉钢,臧埔,等.鹿角脱盘及其5种提取部位21种氨基酸含量对比分析[J].药物分析杂志,2017,37(10):1851-1856.
[17]唐翎,魏伟,赵勇.UPLC-MS/MS法测定猴头菌丝体中18种游离氨基酸的含量[J].药物分析杂志,2018,38(1):112-117.
[18]Le A,Ng A,Kwan T,et al.A rapid,sensitive method for quantitative analysis of underivatized amino acids by liquid chromatography-tandem mass spectrometry(LC-MS/MS).JChromatogr B Analyt Technol Biomed Life Sci.2014;944:166-174.
[19]Roucher VF,Desnots E,Na?l C,et al.Use of UPLC-ESI-MS/MS to quantitate free amino acid concentrations in micro-samples of mammalian milk.Springerplus.2013;2:622.
[20]李泰锋,薛薇,李敏,等.LC-MS/MS法测定大鼠脑透析液中6种氨基酸含量的方法学建立[J].中国新药杂志,2015,24(14):1659-1664.
[21]Zhai XJ,Chen F,Zhu CR,et al.A simple LC-MS/MS method for quantitative analysis of underivatized neurotransmitters in rats urine:assay development,validation and application in the CUMS rat model.Biomed Chromatogr.2015;29(11):1737-1743.
[22]赵芳,李强,梁梅丽.超高效液相色谱-串联质谱法快速测定大鼠血清中8种神经递质[J].分析化学,2018,1(1):121-128.
[23]Kleijkers SH,van Montfoort AP,Bekers O,et al.Ammonium accumulation in commercially available embryo culture media and protein supplements during storage at 2-8°C and during incubation at 37°C.Hum Reprod.2016;6(31):1192-1199.
[2]Petra L,Gardner DK.The effects of chemical and physical factors on mammalian embryo culture and their importance for the practice of assisted human reproduction.Hum Reprod Update.2016;22(1):2-22.
[3]王增艳,何方方,孙正怡,等.冷冻环玻璃化法冷冻小鼠卵母细胞的效果评价[J].生殖医学杂志,2008,17(1):38-42.
[4]李宗武,朱传金,刘庆勇.人血浆及精浆中钙、镁、锌、铜对精液成分的影响[J].国外医学(医学地理分册),2004,25(3):124-126.
[5]Morbeck DE,Krisher RL,Herrick JR,et al.Composition of commercial media used for human embryo culture.Fertil Steril2014;102(3):759-766.
[6]Gardner DK,Vella P,Lane M,et al.Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers.Fertil Steril.1998;69(1):84-88.
[7]Dumoulin JC,Land JA,Van Montfoort AP,et al.Effect of in vitro culture of human embryos on birthweight of newborns.Hum Reprod.2010;25:605-612.
[8]Nelissen EC,Van Montfoort AP,Coonen E,et al.Further evidence that culture media affect perinatal outcome:findings after transfer of fresh and cryopreserved embryos.Hum Reprod.2012;27(7):1966-1976.
[9]赵杰,陈秀娟,范文斌.鼠胚在人类辅助生殖培养环境质量控制的作用研究[J].内蒙古医学院学报,2009,31(1):49-51.
[10]《人类体外辅助生殖技术用液注册技术审查指导原则》http://cnda.cfda.gov.cn/WS04/CL2050/227857.html
[11]EUROPEAN COMMISSION,Guidelines for conformity assessment of In Vitro Fertilisation (IVF) and Assisted Reproduction Technologies(ART)products.[2012-01-01]http://ec.europa.eu/docsroom/documents/10340/attachments/1/.../pdf
[12]Gardner DK,Lane M.Amino acid and ammonium regulate mouse embryo development in culture.Biol Reprod.1993(48):377-385.
[13]Gardner DK,Hamilton R,McCallie B,et al.Human and mouse embryonic development,metabolism and gene expression are altered by an ammonium gradient in vitro.Reproduction.2013;146:49-61.
[14]Hashimoto S,Nishihara T,Murata Y,et al.Medium without ammonium accumulation supports the developmental competence of human embryos.J Reprod Dev.2008;54:370-374.
[15]刘婷婷,邢青斌,程佳丽,等.桦褐孔菌氨基酸含量测定方法的建立[J].营养学报,2017,39(5):504-506.
[16]陈思,郜玉钢,臧埔,等.鹿角脱盘及其5种提取部位21种氨基酸含量对比分析[J].药物分析杂志,2017,37(10):1851-1856.
[17]唐翎,魏伟,赵勇.UPLC-MS/MS法测定猴头菌丝体中18种游离氨基酸的含量[J].药物分析杂志,2018,38(1):112-117.
[18]Le A,Ng A,Kwan T,et al.A rapid,sensitive method for quantitative analysis of underivatized amino acids by liquid chromatography-tandem mass spectrometry(LC-MS/MS).JChromatogr B Analyt Technol Biomed Life Sci.2014;944:166-174.
[19]Roucher VF,Desnots E,Na?l C,et al.Use of UPLC-ESI-MS/MS to quantitate free amino acid concentrations in micro-samples of mammalian milk.Springerplus.2013;2:622.
[20]李泰锋,薛薇,李敏,等.LC-MS/MS法测定大鼠脑透析液中6种氨基酸含量的方法学建立[J].中国新药杂志,2015,24(14):1659-1664.
[21]Zhai XJ,Chen F,Zhu CR,et al.A simple LC-MS/MS method for quantitative analysis of underivatized neurotransmitters in rats urine:assay development,validation and application in the CUMS rat model.Biomed Chromatogr.2015;29(11):1737-1743.
[22]赵芳,李强,梁梅丽.超高效液相色谱-串联质谱法快速测定大鼠血清中8种神经递质[J].分析化学,2018,1(1):121-128.
[23]Kleijkers SH,van Montfoort AP,Bekers O,et al.Ammonium accumulation in commercially available embryo culture media and protein supplements during storage at 2-8°C and during incubation at 37°C.Hum Reprod.2016;6(31):1192-1199.