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闲鹤野云
第12楼2008/02/26
液相色谱-质谱联用技术在测定血液中左炔诺孕酮含量的应用
1.前言
左炔诺孕酮，(17-α)-(+/−)-13-ethyl-17-hydroxy-18,19-dinorpregn-4-en-20-yn-3-one,是一种用于人类预防妊娠的合成类女性避孕激素。对该药物和其他口服避孕药血浆水平的监控，改变了药物配方（降低预期的有效剂量）并从而限制了临床副作用[1]。 Johnston [2]描述了包括左炔诺孕酮在内的避孕药的常规HPLC检测方法。Berzas等[3]评价了毛细管电泳法和HPLC法的异同。但这些方法灵敏性都低，无法满足低剂量左炔诺孕酮药物动力学分析。放免分析方法(RIA)曾在左炔诺孕酮药物动力学分析中使用过，检测限达到pg/mL范围[1,4]。放免分析方法虽然灵敏，但昂贵、费时、具有放射标记危险和非专一性。Lauritsen和Rose [8]描述了使用 desorption chemical ionisation membrane inlet mass spectrometry (DCI-MIMS)包括左炔诺孕酮在内的固醇类物质的鉴别。
1. Introduction
Levonorgestrel, (17-α)-(+/−)-13-ethyl-17-hydroxy-18,19-dinorpregn-4-en-20-yn-3-one, is a synthetic female contraceptive hormone used in pregnancy prevention in humans. The monitoring of plasma levels of this drug as well as other oral contraceptives has brought about changes in formulations (lowering the expected effective dose) and thus limiting clinical side effects [1]. Johnston [2] described
a conventional HPLC method for the determination of oral contraceptives including levonorgestrel. Berzas et al. [3] evaluated the use of capillary electrophoresis versus an HPLC method. These methods are however not sensitive enough for pharmacokinetic analysis of low doses of levonorgestrel. Radio-immuno assay (RIA) methods have been used for determination of levonorgestrel during pharmacokinetic studies [1,4] reaching low detection levels in the pg/ml range. These methods are sensitive, but are expensive, time consuming, hazardous due to radio active labelling and non-specific. Lauritsen and Rose [8], described a method using desorption chemical ionisation membrane inlet mass spectrometry (DCI-MIMS) for identification of steroid hormones including levonorgestrel.
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闲鹤野云
第15楼2008/02/26
LC–MS/MS is becoming the preferred method for quantitative determinations due to rapid, highly selective and sensitive analysis. LC–MS/MS methods using turbo-electrospray ionisation (ESI) for determination of levonorgestrel in human serum [5], environmental water [6] and sewage effluent [7] have been described, but the APPI source used in our laboratory lower the background noise produced by ESI, enabling us to develop a more sensitive method, with high sample throughput due to short chromatographic conditions and simple sample preparation. Kushnir et al. [9] used atmospheric pressure photo-ionisation with tandem mass spectrometry to analyse cortisol and cortisone in serum and plasma.
LC–MS/MS 方法因为其快速、高选择性和灵敏性，正逐渐成为定量分析的优选方法。利用具高速-电喷射离子化技术（turbo-electrospray ionisation (ESI)不懂）的 LC–MS/MS 系统测定人血清、环境水体和污水中左炔诺孕酮已有描述[7]，由于我们实验室使用的APPI源可以降低ESI产生的背景噪音，从而使我们能开发出一个更灵敏的方法。该方法因色谱运行时间短而能快速大量分析样品，制样也简单。Kushnir等[9]利用atmospheric pressure photo-ionisation with tandem mass spectrometry 分析了血清和血浆中的氢化可的松（cortisol）和可的松（ cortisone）的含量。
只能帮你这些了，你自己翻译后大家帮助你修改吧。
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凌云
第18楼2008/03/29
翻译了好几天,大家来补充补充.呵呵
摘要
高效液相色谱-质谱联用技术已经应用于人血中左炔诺孕酮含量的测定。应用生物系统公司的API 3000型三重四极杆质谱应用大气压喷雾离子源，采用多重反应监控（MRM）模式。以17-α-甲基睾酮做内标，用苯基-已基柱进行反相液-液色谱分离，串联质谱进行测定。左炔诺孕酮和17-α-甲基睾酮的平均回收率分别为99.5%和62.9%。每毫升血浆中本方法检测范围为0.265纳克到130纳克，最低检测限为0.265纳克每毫升。本方法使用了高效液相色谱联用高灵敏度和高选择性的质谱技术，测定人血中的左炔诺孕酮。本分析方法研究了健康女性志愿者口服1.5毫克左炔诺孕酮，在五个半衰期内人体血浆中左炔诺孕酮的药物代谢动力学，在本方法中，批次样品的单个样品的色谱分离时间为5分钟。angles-chen 发表:那就分段来吧，呵呵。
1：
Abstract
A selective, sensitive and rapid liquid chromatography–tandem mass spectrometry method for the determination of levonorgestrel in plasma was developed. An Applied Biosystems API 3000 triple quadrupole mass spectrometer set to multiple reaction monitoring (MRM) mode, using atmospheric pressure photospray ionisation (APPI) in the positive mode. Using 17-α-methyltestosterone as internal standard (IS), liquid–liquid extraction was followed by reversed phase liquid chromatography using a phenyl–hexyl column and tandem mass spectrometric detection. The mean recovery for levonorgestrel and 17-α-methyltestosterone was 99.5 and 62.9%, respectively. The method was validated from 0.265 to 130 ng levonorgestrel/ml plasma with the lower limit of quantification (LLOQ) set at 0.265 ng/ml. This assay method makes use of the increased sensitivity and selectivity of tandem mass spectrometric (MS/MS) detection, allowing for a rapid (extraction and chromatography) and selective method for the determination of levonorgestrel in human plasma. The assay method was used in a pharmacokinetic study to quantify levonorgestrel in human plasma samples generated after administrating a single oral dose of 1.5 mg levonorgestrel to healthy female volunteers for up to five half lives. The total chromatographic runtime of this method was 5.0 min per sample, allowing for analysis of a large number of samples per batch.
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凌云
第19楼2008/03/29
2、试验
2.1 试验仪器和试剂
色谱柱：A phenomenex Luna® 苯基-已基反相柱，5μm, 2.0mm×150mm，美国加利福尼亚州，托兰斯，Phenomenex公司；
流速：0.3mL/min低流速；
泵：安捷伦1100型恒流泵，美国加利福尼亚州，Palo Alto，安捷伦公司；
自动进样器：安捷伦1100型自动进样器，美国加利福尼亚州，Palo Alto，安捷伦公司；
检测器：应用生物系统的API 3000型质谱仪，配APPI阳离子源，加拿大，安大略，应用生物系统公司；
乙腈、正己烷、甲醇（Burdick and Jackson高纯度）：美国Baxter化学公司；
蚁酸：英国BDH公司，不用进一步纯化；
异戊醇：德国，Merck公司；
水：经反相渗透微孔过滤；
左炔诺孕酮：法国巴黎Orgasynth Industries ；
17-α-甲基睾酮：FARMOVS-PAREXEL化学合成物研究所。
2.2 试验准备和样品配置
标准溶液的配置：左炔诺孕酮标样用甲醇溶解成备用溶液，用来————。用空白的人体血浆（1：1，V/V）稀释使左炔诺孕酮的含量从0.265至130ng/ml。用同样的方法，用同样的方法配制样品溶液，以不同的备用液稀释使含量从0.340至113ng/ml。标样溶液和样品溶液就配好了。溶液要放在聚丙烯瓶子并在−20℃以下保存。angles-chen 发表:3：
2. Experimental
2.1. Materials and chemicals
A phenomenex Luna® phenyl–hexyl 5μm, 2.0mm× 150mm reverse phase analytical column (Phenomenex, Torrance,CA, USA) was used for chromatographic separation at a flow-rate of 0.3 ml/min. The mobile phase was delivered by an Agilent Series 1100 isocratic pump and the samples injected by an Agilent Series 1100 autosampler (Agilent, Palo Alto, CA, USA). Detection was performed by an Applied Biosystems API-3000 mass spectrometer (Applied Biosystems,Ontario, Canada) fitted with an atmospheric pressure photospray ionisation (APPI) source operating in the positive ion mode.
Acetonitrile, hexane and methanol (Burdick and Jackson,High Purity) were obtained from Baxter chemicals (USA),formic acid from BDH (England) and was used without further purification. Iso-amyl alcohol (pro analysi) was obtained from Merck (Merck, Germany). Water was purified by Millipore Elix 5 reverse osmosis and Milli-Q® (Millipore) Gradient A10 polishing system (Millipore, Bedford, MA, USA).Levonorgestrel (C21H28O2) and 17-α-methyltestosterone (C20H30O2) were obtained from Orgasynth Industries (Paris,France) and FARMOVS-PAREXEL internal chemical reference library, respectively.
2.2. Preparation ofstandar ds and quality control samples
Calibration standards (STD) were prepared by dissolving pure reference standard of levonorgestrel powder in methanol to obtain a stock solution, which was used to spike a pool of blank human plasma (stripped of endogenous components).By serial dilution with blank human plasma (1:1, v/v) a calibration standard range between 0.265 and 130 ng/ml was obtained. Similarly, quality control standards (QC) were prepared (using the same methodology, but different stock solutions) spanning a range between 0.340 and 113 ng/ml.Sufficient calibration standards and quality controls were prepared to validate the method and assay all the study samples.Aliquots of the standards and quality controls were stored in polypropylene tubes together with the study samples at −20℃ until processed.
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凌云
第20楼2008/03/29
2.3 萃取样品
取1ml血样至10ml棕色安踣瓶中，加入100 μl内标溶液（150ng/mL的17-α-甲基睾酮水溶液）和5ml有机溶剂（正己烷：异戊醇，98：2，v/v），摇晃90秒后用离心机在1300×g下使样品分层，水相在−25℃的酒精浴中冷藏，有机相缓慢转移入5 ml棕色安踣瓶中。
有机溶液在55℃下用氮吹仪浓缩至干，剩余物用2%蚁酸溶解，摇晃45秒，混合物放到96孔托盘中放入自动进样器进样。
2.4 液相色谱条件
流动相：乙腈：甲醇：0.1%蚁酸=45：35：20；
流量：0.3ml/min；
所有色谱试剂都在氦气流中脱气4分钟。
2.5 质谱条件
大气压光电离离子源在阳离子模式下运行 热石英管温度设置在380℃。
后面不会了angles-chen 发表:4：
2.3. Extraction procedure
Plasma samples (1 ml) were pipetted into 10 ml amber ampoules, 100 μl internal standard solution (150 ng 17-α-methyltestosterone/ml water) and 5 ml organic solvent (hexane:iso-amyl alcohol, 98:2, v/v) added. The samples were vortex-mixed for 90 s and centrifuged at 1300×g to aid layer separation of the aqueous and organic solvents.The aqueous phase was frozen in an alcohol freezing bath at −25℃ and the organic layer was decanted into a 5 ml amber ampoule.
The organic solvent was evaporated under a stream of nitrogen at 55℃ until dry. The residue was reconstituted in 150μl, 2% formic acid solution and vortexed for 45 s. The mixture was transferred to 96 deep-well plates, placed onto the autosampler and injected.
2.4. Liquid chromatography
Chromatography was performed at ambient temperature with a mobile phase consisting of acetonitrile, methanol, and 0.1% formic acid (45:35:20, v/v/v) at a flow-rate of 0.3 ml/min. All chromatographic solvents were degassed by sparging helium through the solution for 4 min.
2.5. Mass spectrometry
Atmospheric pressure photospray ionisation was performed in the positive ion mode with nitrogen as the nebulizing and auxiliary gas both set at an optimal value of 9 (arbitrary values) and the temperature of the heated quartz tube was set at 380℃. The APPI source settings were obtained after conducting flow injection analysis. Toluene was used as a dopant-liquid and delivered via a post column APPI source interface T-piece at a flow rate of 30 μl/min. A turbo electrospray ionisation (ESI) source was used to optimise the triple quadrupole settings of the instrument for detection of levonorgestrel and 17-α-methyltestosterone by infusing a 500 ng/ml solution of each drug dissolved in a formic acid: methanol (1:99, v/v) solution at a constant flow rate of 10μl/min. The pause time was set at 5ms and the dwell time at 150 ms. The collision gas (N2) was set at 9 (arbitrary value).
The Applied Biosystems API 3000 mass spectrometer was operated at unit resolution in the multiple reaction monitoring (MRM) mode, monitoring the transition of the protonated molecular ions m/z 313.0 and 303.0 to the product ions m/z 109.1 and 96.9 for levonorgestrel and 17-α-methyltestosterone, respectively. Fig. 1 shows a full scan mass spectrum of pure levonorgestrel showing the M+ 1 precursor ion of protonated levonorgestrel (m/z 313.0, molecular structure given) overlaid by a full-scan mass spectrum of levonorgestrel after collision, showing the most abundant product ions and the principal product ion at m/z 109.1. Fig. 2 shows a full scan mass spectrum of pure 17-α-methyltestosterone showing the M+ 1 precursor ion of protonated 17-α-methyltestosterone (m/z 303.0, molecular structure given) overlaid by a full scan mass spectrum of 17-α-methyltestosterone after collision, showing the most abundant product ions and the principal product ion at m/z 96.9. The instrument was interfaced with a computer running Applied Biosystems Analyst version 1.2 software.
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凌云
第21楼2008/03/29
在甲醇溶液中，左炔诺孕酮在4℃, −20℃和室温下放置24小时以上不降解；在血样中，左炔诺孕酮在室温下放置16小时以上不降解。
不同浓度的左炔诺孕酮存放在聚丙烯瓶子中，于−20℃经过267天的稳定性考察，结果表明此条件下左炔诺孕酮是稳定的。
4 结论
介绍了一个用高效液相色谱分离，串联质谱法快速、灵敏、高选择性测定人血中左炔诺孕酮的的方法。大气压光电离离子源(APPI+)的灵敏度是 离子源(ESI+)的四倍。大气压光电离离子源的优势使运用液-质联用法高灵敏度地测定药物代谢动力学时大量样品成为可能。此新方法研究了18位口服1.5mg左炔诺孕酮的更年期女性志愿者的药物代谢，选择性比以前报道的方法（HPLC，TLC和RIA）更好，色谱时间也更短（5分钟），样品制备也更简单。高性能的液-质联用手段用来检测批次中的微小变化。色谱分析柱在整个分析过程中足以把样品清洗干净，因此在研究过程中没有必要清洗离子源。本方法在分析人血中的左炔诺孕酮是一个不错的方法。angles-chen 发表:7：
Levonorgestrel is stable in solution (methanol) at 4℃, −20℃ and room temperature for at least 24 h. No degradation occurred after leaving quality control plasma samples on bench top at room temperature over a period of 16 h.
Long term matrix stability at −20℃ was assessed over a period of 267 days using two different concentrations of levonorgestrel. Levonorgestrel is stable in plasma when stored at −20℃ in polypropylene tubes for at least 267 days (Table 4).
4. Conclusion
A rapid, sensitive and highly selective method for the determination of levonorgestrel in plasmawas developed, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. Atmospheric pressure photospray ionisation (APPI+) improved the sensitivity four times compared to turbo-electrospray ionisation (ESI+). The advantages of using atmospheric pressure photospray ionisation made it possible to use this LC–MS/MS method for analysis of large number of samples with great precision during pharmacokinetic studies. This newly developed assay method was used in a pharmacokinetic study in which 18 healthy post menopause female volunteers were each given a 1.5 mg single oral dose of levonorgestrel. The assay method is more selective than previously described methods (HPLC, TLC and RIA) and allows for a much higher sample throughput due to the short chromatography time (5.0 min) and simple sample preparation. Robust LC–MS/MS instrument performance was observed, with only slight variations in the instrument response within batches. It was not necessary to clean the ion source during the entire study.Asingle analytical columnwas used to chromatograph about 1250 extracts and was still in good working condition after the study was completed indicating sufficient sample clean-up. This method is an excellent analytical option for rapid quantification of levonorgestrel in human plasma.