反相离子对高效液相色谱分析~(125)Ⅰ-甲状腺激素

甲状腺激素3,5,3′,5′-四碘甲腺原氨酸(T_4),3,5,3′-三碘甲腺原氨酸(T_3)及3,3′,5′-三碘甲腺原氨酸(γT_3)为甲状腺碘代氨基酸。它们的~(125)I标记化合物分别为T_3、T_4、γT_3的碘-125标记药盒的主要组成。这些药盒分别用于测定血清中T_3,T_4,γT_3的浓度,以判断甲状腺对组织的成熟、生长、代谢活动和功能,诊断甲状腺疾病。     

高效液相色谱-串联质谱检测牛奶中的甲状腺素

建立了高效液相色谱-串联质谱(HPLC-MS/MS)检测牛奶中甲状腺素3,3′,5,5′-四碘-L-甲腺原氨酸(T4), 3,3′,5-三碘-L-甲腺原氨酸(T3)和3,3′,5′-三碘-L-甲腺原氨酸(rT3)的方法。样品用乙腈提取,离心,上清液经氨水碱化和Cleanert PAX固相萃取小柱净化,在Zorbax Eclipse XDB-C18 (150 mm×2.1 mm, 3.5 μm)色谱柱上以0.1%(v/v)乙酸水溶液和甲醇为流动相等度洗脱分离,以电喷雾正离子(ESI+)模式电离,多反应监测(MRM)模式质谱检测,内标法定量。结果表明,甲状腺素的检出限(LOD)不大于0.03 ng/g,定量限(LOQ)不大于0.1 ng/g;在考察的浓度范围内线性关系良好(r2＞ 0.998);回收率为80.61%～101.7%,相对标准偏差(RSD)为1.48%～9.70%。室温下样品溶液中的甲状腺素保持稳定。对5个牛奶样品的测定结果显示,T3含量为0.59～1.30 ng/g, RSD为2.06%～7.70%; T4和rT3未检出。该方法具有样品处理简单,灵敏度高,重现性好,定性和定量结果可靠等特点,为牛奶中甲状腺素的测定和相关质量安全评价提供了可靠手段。     

基于3,5-二胺基-N（N′,N′-正二丁基甲酰胺基苯基）-苯甲酰胺的聚酰亚胺的溶解性、热稳定性和取向性能

用制得的大体积双侧基3,5-二胺基-N（N′,N′-正二丁基甲酰胺基苯基）-苯甲酰胺（DAPDM）,与3,3′-二甲基-4,4′-二氨基二苯甲烷（DMMDA）、3,3′,4,4′-二苯醚四甲酸二酐（ODPA）采用一步法共缩聚制得聚酰亚胺共聚物。采用核磁共振对该共聚物进行了表征,对其溶解性、热稳定性、预倾角进行了分析。结...     

具有甲状腺素脱碘酶活性的抗体酶的化学修饰

甲状腺素脱碘酶是一种膜硒蛋白[1], 它能够将甲状腺素降解为不同产物, 并对甲状腺素的生理功能起调控作用. 但是, 甲状腺素脱碘酶极易变性失活, 到目前为止仍未得到纯酶[2]. 近来, 此酶的人工模拟工作逐渐成为热点. 我们小组[3]首次以3,5,3′,5′-四碘甲状腺原氨酸五水钠盐(T4)为半抗原, 采用单克隆抗体技术和苯甲基磺酰氟及硒氢化钠修饰的方法, 成功地制备了具有甲状腺素脱碘酶活性的抗体酶.     

液相色谱/质谱法分离鉴定碘化酪蛋白水解产物中的碘化氨基酸

用Waters Symmetrys Shield^TM RP18柱，乙腈(B，含0．1％甲酸)和水(A，含0．1％甲酸)为流动相，0．8mL／min，线性洗脱(B从10％到50％)40min，液质联用(LC／MS)，电喷雾电离源(ESI^ ，SIR方式)，对碘化酪蛋白水解物中的一碘酪氨酸(MTT)、二碘酪氨酸(DIT)、二碘甲腺原氨酸(T2)、三碘甲腺原氨酸(L3,rT3)和四碘甲腺原氨酸(T4)进行了分离确证。同时测定了T3、rT3和T4的含量。     

碘酸根与3,3‘,5,5’—四甲基联苯胺的显色反应研究及其分析应用

研究了碘酸根与 3,3′,5 ,5′-四甲基联苯胺 (TMB)的显色反应 ,建立了测定微量碘的分光光度法。在盐酸介质中 ,碘酸根能定量氧化 TMB生成黄色的TMB亚胺盐 ,其最大吸收波长为 45 0 nm,表观摩尔吸光系数为 2 .13× 10 5 L·mo1-1·cm-1 ,碘的质量浓度在 0～ 0 .7mg/ L 范围内符合比尔定律 ,该法已应用于加碘酸钾食盐中碘含量的测定     

高速逆流色谱分离纯化九里香中的黄酮类化合物

应用高速逆流色谱法分离纯化了九里香中的4种黄酮类化合物。以石油醚-乙酸乙酯-甲醇-水(5:5:4.8:5, v/v/v/v)作为两相溶剂系统,上相为固定相,下相为流动相,以主机转速800 r/min、流速2.0 mL/min、单次进样量200 mg的条件成功地从4.0 g九里香粗提物中分离纯化出54.31 mg 5,7,3′,4′,5′-五甲氧基黄酮(重结晶后)、107.68 mg 5-羟基-6,7,3′,4′-四甲氧基黄酮、215.54 mg 5-羟基-6,7,8,3′,4′-五甲氧基黄酮、84.36 mg 5-羟基-6,7,8,3′,4′,5′-六甲氧基黄酮,纯度均在95%以上。各化合物的结构均由质谱和核磁共振氢谱、碳谱鉴定。其中化合物5-羟基-6,7,3′,4′-四甲氧基黄酮为首次从九里香中分离得到。     

从中药佛手中分离鉴定两个柠檬油素二聚体

用 IR、NMR和X-射线晶体衍射分析了从佛手中分离出来的两个柠檬油素二聚体,证明它们的化学结构分别是顺式头-尾-3,3′,4,4′-柠檬油素二聚体(1)和顺式头-头-3,3′,4,4′-柠檬油素二聚体(2)。通过光反应从柠檬油素制备了1和反式头-头-3,3′,4,4′-柠檬油素二聚体(3)。     

柱前手性荧光衍生反相高效液相色谱法分离甲状腺素对映体

以R(-)-4-N,N-二甲基磺酰胺-7-(3-异氰酸吡咯烷)-2,1,3-苯并氧杂咪唑（R(-)-DBD-PyNCS）为手性荧光衍生化试剂,成功地拆分了甲状腺素对映体D,L-四碘甲状腺原氨酸（T4）和L-三碘甲状腺原氨酸(T3)。在反应温度为40 ℃、反应时间为20 min时,R(-)-DBD-PyNCS在碱性介质中可与甲状腺素对映体生成稳定的非对映体衍生物。该衍生物在以乙腈-水-醋酸(体积比为60∶40∶1)为流动相,流速为1.0 mL/min,色谱柱为Intersil-ODS-3 C18柱(150 mm×4.6 mm,5 μm)的色谱条件下得到了充分的分离。采用荧光检测器在激发波长460 nm、发射波长550 nm下检测。D,L-T4和L-T3分别在0.016～0.30 μg/μL和0.0067～0.22 μg/μL范围内,峰面积与浓度呈良好的线性关系(r＞0.999)。D,L-T4和L-T3的最低检出限分别为0.02 μg/mL和0.85 μg/mL(S/N=3)。在D-T4、L-T4、L-T3质量浓度分别为0.10 μg/μL下测得峰面积的相对标准偏差分别为3.40%,1.63%,3.30%(n=7)。该方法成功地应用于甲状腺片中T4和T3的含量测定。     

5,5′-二甲基-2,2′-二茚满-1,1′,3,3′-四酮的合成及结构表征

2,2′-二茚满-1,1′,3,3′-四酮(1)是合成茚满类光致磁性材料[1]的重要中间体, 在军事目标识别、特殊防伪以及光信息存储等领域具有潜在的应用价值, 因此取代的2,2′-二茚满-1,1′,3,3′-四酮的合成已成为人们研究的焦点.     

Differentiation of diiodothyronines using electrospray ionization tandem mass spectrometry

Diiodothyronines 3,5-diiodothyronine (3,5-T2), 3',5'-diiodothyronine (3',5'-T2), and 3,3'-diiodothyronine (3,3'-T2) are important metabolites of 3,5,3'-triiodothyronine (T3) and 3,3',5'-triiodothyronine (rT3; reverse T3). In this paper, a novel and rapid method for identifying and quantifying 3,5-T2, 3',5'-T2 and 3,3'-T2 has been introduced using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Fragmentation patterns were proposed on the basis of our data obtained by ESI-MS/MS. MS2 spectra in either negative ionization mode or positive ionization mode can be used to differentiate 3,5-T2, 3',5'-T2 and 3,3'-T2. On the basis of the relative abundance of fragment ions in MS2 spectra under the positive ionization mode, quantification of the 3,5-T2, 3',5'-T2 and 3,3'-T2 isomers in mixtures is also achieved without prior separation.     

Simultaneous quantification of T4, T3, rT3, 3,5‐T2 and 3,3′‐T2 in larval zebrafish (Danio rerio) as a model to study exposure to polychlorinated biphenyls

Environmental toxicants that interfere with thyroid hormone (TH) signaling can impact growth and development in animals and humans. Zebrafish represent a model to study chemically induced TH disruption, prompting the need for sensitive detection of THs. Simultaneous quantification of 3,3′,5‐triiodo‐l ‐thyronine (T3), thyroxine (T4), 3,3′,5′‐triiodo‐l ‐thyronine (rT3), 3,5‐diiodo‐l ‐thyronine (3,5‐T2) and 3,3′‐diiodo‐l ‐thyronine (3,3′‐T2) in zebrafish larvae was achieved by ultra‐performance liquid chromatography–tandem mass spectrometry in positive ion mode. Solid‐phase extraction with SampliQ cartridges and derivatization with 3 m hydrochloric acid in n‐butanol reduced matrix effects. Derivatized compounds were separated on an Acquity UPLC BEH C₁₈ column with mobile phases consisting of 0.1% acetic acid in deionized water and 0.1% acetic acid in methanol. The limits of detection ranged from 0.5 to 0.6 pg injected on column. The method was validated by evaluating recovery (77.1–117.2%), accuracy (87.3–123.9%) and precision (0.5–12.4%) using diluted homogenized zebrafish embryos spiked with all target compounds. This method was then applied to zebrafish larvae collected after 114 h of exposure to polychlorinated biphenyls (PCBs), including PCB 28, PCB 66 and PCB 95, or the technical mixture Aroclor 1254. Exposure to PCB 28 and PCB 95 increased the T4:T3 ratio and decreased the T3:rT3 ratio, demonstrating that this method can effectively detect PCB‐induced alterations in THs.     

Determination of ethyl-glucuronide in hair for heavy drinking detection using liquid chromatography-tandem mass spectrometry following solid-phase extraction

The detection of ethyl-β-d-6-glucuronide (EtG), a stable phase II metabolite of ethanol, is of interest in both clinical and forensic contexts with the aim of monitoring alcohol abuse. We present a liquid chromatography-electrospray ionisation-tandem mass spectrometry method for the detection and quantification of EtG in hair. Thirty milligrams of washed and cut hair were cleaned up using solid-phase extraction graphite cartridges. Separation was then performed using an Uptisphere-3SI column, and the detection was operated in the negative mode. After validation, the method was applied to hair samples taken from four fatalities (F) with documented excessive drinking habits, 12 heavy drinkers (HD) and seven social drinkers (SD). The method exhibits limits of detection and quantification of 4 and 10 pg/mg, respectively. Intra- and inter-assay standard deviation and relative bias were less than 20% over the calibrating range (10 to 3,000 pg/mg). EtG hair concentrations in SD were <10 pg/mg and >50 pg/mg for F and HD (range, 54 to 497 pg/mg). The present assay appears convenient to carry out owing to the very small quantity of hair samples required to determine an effective heavy alcohol consumption (EtG hair concentration >50 pg/mg).     

头发中内源性类固醇激素的气相色谱-串联质谱分析

建立了建康人头发中内源性类固醇兴奋剂睾酮、表睾酮、雄酮、苯胆烷醇酮和脱氢表雄酮的气相色谱-串联质谱(GC-MS/MS)分析方法。头发经碱水解后,以乙醚提取,经衍生化后采用GC-MS/MS的多反应监测模式(MRM)分析。方法的线性关系良好,检出限达0.1～0.2 pg/mg;提取回收率为74.6%～104.5%;日内测定的准确度为90.1%～113.7%,日内及日间测定的精密度均小于17.5%。应用所建立的方法测定了80例中国健康人头发中睾酮、表睾酮、雄酮、苯胆烷醇酮和脱氢表雄酮的生理水平,为内源性类固醇兴奋剂滥用的判断提供了方法和基础数据。     

Regioselective deiodination of thyroxine by iodothyronine deiodinase mimics: an unusual mechanistic pathway involving cooperative chalcogen and halogen bonding

Iodothyronine deiodinases (IDs) are mammalian selenoenzymes that catalyze the conversion of thyroxine (T4) to 3,5,3'-triiodothyronine (T3) and 3,3',5'-triiodothyronine (rT3) by the outer- and inner-ring deiodination pathways, respectively. These enzymes also catalyze further deiodination of T3 and rT3 to produce a variety of di- and monoiodo derivatives. In this paper, the deiodinase activity of a series of peri-substituted naphthalenes having different amino groups is described. These compounds remove iodine selectively from the inner-ring of T4 and T3 to produce rT3 and 3,3'-diiodothyronine (3,3'-T2), respectively. The naphthyl-based compounds having two selenols in the peri-positions exhibit much higher deiodinase activity than those having two thiols or a thiol-selenol pair. Mechanistic investigations reveal that the formation of a halogen bond between the iodine and chalcogen (S or Se) and the peri-interaction between two chalcogen atoms (chalcogen bond) are important for the deiodination reactions. Although the formation of a halogen bond leads to elongation of the C-I bond, the chalcogen bond facilitates the transfer of more electron density to the C-I σ* orbitals, leading to a complete cleavage of the C-I bond. The higher activity of amino-substituted selenium compounds can be ascribed to the deprotonation of thiol/selenol moiety by the amino group, which not only increases the strength of halogen bond but also facilitates the chalcogen-chalcogen interactions.     

Glyphosate and aminomethylphosphonic acid in human hair quantified by an LC-MS/MS method

An LC-MS/MS method for hair testing of glyphosate and aminomethylphosphonic acid (AMPA), its main biodegradation product, has been developed. After decontamination, 50 mg of hair was ground and sonicated in water for 2 h. The method was fully validated in the 5–500 pg/mg range for glyphosate and 10–500 pg/mg for AMPA, and the limits of detection were 2 and 5 pg/mg, respectively. Matrix effect for glyphosate and AMPA was compensated by an isotope-labeled internal standard. Hair samples from four farmers who regularly used glyphosate and one farmer who used glyphosate but not his wife and 14 hair samples from nonoccupationally exposed subjects were tested. Glyphosate was found in head hair of three farmers, with concentration in the range 14–188 pg/mg. The fourth was found negative but with hair colored in red. Glyphosate was detected in 10 of 14 hair samples from nonoccupationally exposed subjects at concentrations of 11.5 pg/mg or lower and only in one segment (0–3 cm) of the farmer's spouse (6 pg/mg). AMPA was detected in five subjects, above the limit of quantification only in two of three occupationally exposed subjects with positive glyphosate. Further studies should be conducted to validate this potential new biomarker that could be useful for assessing long-term exposure to glyphosate.     

Differentiation of monoiodothyronines using electrospray ionization tandem mass spectrometry

In this work two monoiodothyronines, 3-T1 and 3'-T1, have been analyzed using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Fragmentation patterns were proposed based on our data obtained by ESI-MS/MS. MS2 spectra in either negative or positive ion mode can be used to differentiate 3-T1 and 3'-T1. Based on the relative abundance of fragment ions in MS2 spectra in the negative ion mode, quantification of the 3-T1 and 3'-T1 isomers in mixtures is achieved without prior separation. Solid-phase extraction in combination with ESI-MS/MS provides a practicable procedure that can be used to determine the molar ratio of 3-T1 and 3'-T1 in human serum with an error less than 3%. The detection limits for 3-T1 and 3'-T1 were 0.5 and 0.7 pg/microL, respectively.     

Development and validation of an LC‐MS/MS method for quantification of Δ9‐tetrahydrocannabinolic acid A (THCA‐A), THC,CBN and CBD in hair

For analysis of hair samples derived from a pilot study (‘in vivo’ contamination of hair by sidestream marijuana smoke), an LC‐MS/MS method was developed and validated for the simultaneous quantification of Δ9‐tetrahydrocannabinolic acid A (THCA‐A), Δ9‐tetrahydrocannabinol (THC), cannabinol (CBN) and cannabidiol (CBD). Hair samples were extracted in methanol for 4 h under occasional shaking at room temperature, after adding THC‐D₃, CBN‐D₃, CBD‐D₃ and THCA‐A‐D₃ as an in‐house synthesized internal standard. The analytes were separated by gradient elution on a Luna C18 column using 0.1% HCOOH and ACN + 0.1% HCOOH. Data acquisition was performed on a QTrap 4000 in electrospray ionization‐multi reaction monitoring mode. Validation was carried out according to the guidelines of the German Society of Toxicological and Forensic Chemistry (GTFCh). Limit of detection and lower limit of quantification were 2.5 pg/mg for THCA‐A and 20 pg/mg for THC, CBN and CBD. A linear calibration model was applicable for all analytes over a range of 2.5 pg/mg or 20 pg/mg to 1000 pg/mg, using a weighting factor 1/x. Selectivity was shown for 12 blank hair samples from different sources. Accuracy and precision data were within the required limits for all analytes (bias between ?0.2% and 6.4%, RSD between 3.7% and 11.5%). The dried hair extracts were stable over a time period of one to five days in the dark at room temperature. Processed sample stability (maximum decrease of analyte peak area below 25%) was considerably enhanced by adding 0.25% lecithin (w/v) in ACN + 0.1% HCOOH for reconstitution. Extraction efficiency for CBD was generally very low using methanol extraction. Hence, for effective extraction of CBD alkaline hydrolysis is recommended. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of ethyl glucuronide in hair samples by liquid chromatography/electrospray tandem mass spectrometry

A method for the determination of ethyl glucuronide (EtG) in hair samples, using liquid chromatography/electrospray tandem mass spectrometry (LC/ESI-MS/MS), was developed and validated. The treatment of hair samples was as follows: to 100 mg of washed (dichloromethane followed by methanol, 1 ml each) and cut (1-2 mm) material, 700 microl of water, 20 microl of internal standard solution (pentadeuterated EtG, D(5)-EtG, 500 microg/l) and 20 microl of methanol were added. Samples were incubated at 25 degrees C overnight and then ultrasonicated for 2 h. Finally, 8 microl of the centrifuged solution (13,000 rpm) were analyzed by LC/ESI-MS/MS in negative ion mode. The surviving ions of EtG and D(5)-EtG were monitored together with the following MRM transitions: m/z 221 --> 75, m/z 221 --> 85 (EtG) and m/z 226 --> 75, m/z 226 --> 85 (D(5)-EtG). The method exhibited a mean correlation coefficient better than 0.9998 over the dynamic range (3-2000 pg/mg). The lower limit of quantification (LLOQ) and the limit of detection (LOD) were 3 and 2 pg/mg respectively. The intra- and interday precision and accuracy were studied at four different concentration levels (3, 5, 56 and 160 pg/mg) and were always better than 7% (n = 5). Matrix effects did not exceed 20%. The method was applied to several hair samples taken from autopsies of known alcoholics, from patients in withdrawal treatment, from social drinkers, from adult teetotalers and from children not exposed to ethanol, with EtG concentrations globally ranging from < or =2 to 4180 pg/mg.