Determination of IGF-I in horse plasma by LC electrospray ionisation mass spectrometry

The insulin-like-growth factor (IGF-I) peptide is considered to be the main indirect marker for growth hormone administration (GH) in a horse. Further to a previous investigation on measurement of IGF-I in plasma samples by mass spectrometry, this study focuses on quantitative and qualitative analysis of intact IGF-I in horse plasma. First, protein-transposing software has been developed for IGF-I to facilitate its quantification by HPLC–electrospray–ion-trap mass spectrometry. Second, product-ion scan experiments on IGF-I have been conducted on standard samples, non-fortified equine plasma samples, fortified plasma samples, and equine GH post-administration samples. This “top-down” approach method enables characterisation of fragment ions corresponding to the carboxy terminal end, which can be useful for the confirmation of the presence of IGF-I in plasma samples. Figure Structure of IGF-I and amino acid sequences of IGF-I and R3 IGF-I. Deconvolution mass spectra of the IGF-I and R3 IGF-I mixture     

Removal of isobaric interference using pseudo-multiple reaction monitoring and energy-resolved mass spectrometry for the isotope dilution quantification of a tryptic peptide

Energy-resolved mass spectrometry (ERMS) and an isotopically labelled internal standard were successfully combined to accurately quantify a tryptic peptide despite the presence of an isobaric interference. For this purpose, electrospray ionisation tandem mass spectrometry (ESI-MS/MS) experiments were conducted into an ion trap instrument using an unconventional 8 m/z broadband isolation window, which encompassed both the tryptic peptide and its internal standard. Interference removal was assessed by determining an excitation voltage that was high enough to maintain a constant value for the analyte/internal standard peaks intensity ratio, thus ensuring accurate quantification even in the presence of isobaric contamination. Pseudo-multiple reaction monitoring (MRM) was employed above this excitation voltage to quantify the trypic peptide. The internal standard calibration model showed no lack of fit and exhibited a linear dynamic range from 0.5 μM up to 2.5 μM. The detection limit was 0.08 μM. The accuracy of the method was evaluated by quantifying the tryptic peptide of three reference samples intentionally contaminated with the isobaric interference. All the reference samples were accurately quantified with ∼1% deviation despite the isobaric contamination. Furthermore, we have demonstrated that this methodology can also be applied to quantify the isobaric peptide by standard additions down to 0.2 μM. Finally, liquid chromatography ERMS (LC ERMS) experiments yielded similar results, suggesting the potential of the proposed methodology for analysing complex samples.     

Rapid finding and quantification of the major antioxidant in water extracts of three marine drug organisms from China by online HPLC-DAD/MS-DPPH

A method based on high-performance liquid chromatography (HPLC) with diode array detector coupled with electrospray ionisation-mass spectrometry and an online detection system for radical scavenging was established and used to rapidly find and quantify antioxidant compounds in the water extracts of Hippocampus japonicus Kaup, Hippocampus kuda Bleeker and Syngnathus acus Linnaeus. The online screening results revealed the presence of one major radical scavenging compound identified as hypoxanthine by comparison of mass data and retention time with the standard. Subsequently, the developed HPLC method was applied to quantify hypoxanthine in different H. japonicus, H. kuda and S. acus samples. The results indicated that the developed HPLC method is simple and reliable for the quantification of hypoxanthine with a detection limit at 0.002 μg mL(-1), and a high recovery from 96.3% to 102.1%. This method provides a powerful tool for rapid identification and quantification of free radical scavenging compounds in complex marine natural products.     

Determination of limonoate and nomilinoate A-ring lactones in citrus juices by liquid chromatography-electrospray ionization mass spectrometry

The development of delayed bitterness in citrus products is a major problem to citrus producers and juice processors worldwide. A rapid and sensitive liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method has been developed to quantify the recognized precursors of limonoid derived delayed bitterness, limonoate and nomilinoate A-ring lactones, in a wide variety of citrus juices. The limonoid A-ring lactones were isolated by solid-phase extraction from juice samples, analyzed by negative ion LC-ESI-MS and quantified utilizing the standard addition method.     

气相色谱-三重四极杆质谱法同时测定烟丝中73种香气物质

建立了气相色谱-三重四极杆质谱(GC-MS/MS)同时测定烟丝中73种香气物质的分析方法。样品用无水乙醚振荡提取,提取液经过滤后直接进入DB-5MS色谱柱(30 m×0.25 mm,0.25μm)分离,通过优化质谱参数,可有效降低复杂基质和重叠峰的干扰,同时采用多反应监测(MRM)模式测定,内标法定量。结果表明,目标物在各自的线性范围内线性关系良好(r~20.99),低、中、高3个水平的加标回收率为70.0%~122.3%,相对标准偏差(RSD)为1.6%~20.4%。该方法具有前处理简单、准确灵敏的特点,适用于烟丝样品中73种香气物质的检测。     

Quantification of paraquat in postmortem samples by gas chromatography-ion trap mass spectrometry and review of the literature

Paraquat (PQ) is an herbicide implicated in numerous fatalities, mainly caused by voluntary ingestion. Several methods have been used to quantify PQ in plasma and urine samples of intoxicated humans as a predictor of clinical outcome. There is no validated method for the analysis of PQ in postmortem samples. Therefore, the aim of this study was to develop an analytical method, using gas chromatography–ion trap mass spectrometry (GC‐IT/MS) after solid‐phase extraction, to quantify PQ in postmortem samples, namely in whole blood, urine, liver, lung and kidney, to cover the routes of distribution, accumulation and elimination of PQ. The method proved to be selective as there were no interferences of endogenous compounds with the same retention time as PQ and ethyl paraquat (internal standard). The regression analysis for PQ was linear in the range 0–10 µg/mL. The detection limits ranged from 0.0076 µg/mL for urine to 0.047 µg/mL for whole blood, and the recoveries were suitable for forensic analysis. The proposed GC‐IT/MS method provided an accurate and simple assay with adequate precision and recovery for the quantification of PQ in postmortem samples. The proof of applicability was performed in two fatal PQ intoxications. A review of the analytical methods for the determination of quaternary ammonium herbicides is also provided for a better understanding of the presently available techniques. Copyright © 2011 John Wiley & Sons, Ltd.     

自动石墨消解–电感耦合等离子体质谱法同时测定左氧氟沙星胶囊中7种金属元素

建立自动石墨消解-电感耦合等离子体质谱法(ICP-MS)同时测定左氧氟沙星胶囊中铅、铬、砷、镉、锡、铝、铁7种金属元素含量的方法。以HNO3-H2O2()体积比为1∶1为消解体系,采用自动石墨消解法消解左氧氟沙星胶囊样品,消解液除酸后,用5%硝酸溶液定容至50 mL,采用电感耦合等离子体质谱法对消解液进行测定,以内标法定量。铅、铬、砷、镉、锡、铝、铁的质量浓度在0.05~20.0μg/mL范围内与质谱响应值成良好的线性关系,相关系数均大于0.998,方法检出限为0.119~1.323μg/kg。样品加标回收率为91.2%~105.5%,测定结果的相对标准偏差为1.67%~3.46%(n=6)。该方法样品前处理简单,检出限低,测定结果准确,适用于左氧氟沙星胶囊等沙星类抗生素中多种金属元素残留的测定。     

LC–APCI–MS Quantitative Analysis of Curcumin in Rabbit Plasma

A simple and specific liquid chromatography–atmospheric pressure chemical ionization–mass spectrometry (LC–APCI–MS) method was developed and validated for quantitative analysis of curcumin in rabbit plasma. Chromatographic separation was carried out on Zorbax SB-C18 column, acetonitrile-methanol–water used as mobile phase with gradient elution. APCI source was applied and operated in negative ion mode, and selective ion monitoring mode used to quantify curcumin. The method showed excellent intra-assay and inter-assay precision (relative standard deviations and bias <15%) for quality control (QC) samples. This method was successfully applied to a pharmacokinetic study of curcumin after intravenous administration to rabbits.     

Electrochemical Determination of the Antioxidant Activity in Echinacea Purpurea Roots Using Square Wave Voltammetry

A simple and rapid electrochemical method was established to quantify the total polyphenol (TP) content and assess their antioxidant activity (AA) in roots of three Echinacea purpurea (E. purpurea ) species using square wave voltammetry (SWV). Individual polyphenol components were identified, and then quantified by ultra‐high performance liquid chromatography coupled with mass spectrometery (UPLC‐MS). Two major polyphenols, chicoric (ChA) and caftaric (CFT) acids, were identified by mass spectroscopy in the extract of E. purpurea samples. The Accuracy of the proposed SWV electrochemical method for TP content and AA analysis was validated by the highly sensitive UPLC‐MS technique and standard ABTS method, respectively. A high correlation was noticed between the results, indicating the high sensitivity and reliability of the proposed SWV method for polyphenols analysis and AA evaluation in natural herbal samples.     

Liquid chromatography/tandem triple-quadrupole mass spectrometry for determination of paclitaxel in rat tissues

A liquid chromatography/tandem triple-quadrupole mass spectrometry assay to quantify paclitaxel in rat tissue homogenates containing taxol or paclitaxel nanoliposome (PTX-NLP) was developed and validated. Liquid-liquid extraction with tert-butyl methyl ether was used for tissue sample preparation and docetaxel was used as the internal standard. Paclitaxel and docetaxel were separated on a 200 mm x 4.6 mm x 5 microm C(18) column and quantified using a triple-quadrupole mass spectrometer operating in positive ion electrospray selective reaction monitoring mode (ESI(+)-SRM) with a total run time of 6.0 min. The peak area of the m/z 876.3 --> 307.9 transition of paclitaxel is measured versus that of the m/z 830.3 --> 549.1 transition of docetaxel to generate the standard curves. The standard curves were linear over the concentration range of 0.2008-2008 ng/mL for different tissues. The method had high extraction recovery (>90%) and accuracy (>90%) with the intra-day and inter-day precision <15%. Frozen stability, freeze/thaw stability, extraction stability and solution stability at ambient temperature were examined, which indicated the tissue samples should be extracted within 5 days and avoid being frozen and thawed repeatedly over 5 times. Extracted samples after evaporation could be stored at -20 degrees C for 20 days without drug degradation and no degradation was also observed after solution samples were left to stand at ambient temperature for 24 h. This assay was used to support an in vivo biodistribution study of PTX-NLP in rats.     

Determination of the lipid peroxidation product trans-4-hydroxy-2-nonenal in biological samples by high-performance liquid chromatography and combined capillary column gas chromatography-negative-ion chemical ionisation mass spectrometry

trans-4-Hydroxy-2-nonenal (HNE) is an aldehyde end-product of lipid peroxidation in biological systems which is capable of producing a range of powerful biological effects. We wish to describe a sensitive and selective strategy for the determination of HNE in biological samples. The method is based on the formation of the O-pentafluorobenzyl (O-PFB) oxime derivatives of HNE and its deuterated internal standard which, after sample clean-up by solid-phase extraction and purification by high-performance liquid chromatography (HPLC), were derivatised further to trimethylsilyl ethers. Subsequent capillary column gas chromatography-negative-ion chemical ionisation mass spectrometry (GC-NICIMS) using selected-ion monitoring allowed quantitation in the low ng/ml range. The use of an internal standard and the O-PFB oxime derivatives circumvented the problems encountered previously by other workers because of the volatility and instability of HNE. The syn-isomer of HNE O-PFB oxime followed the anti-isomer on the HPLC and GC columns used, giving a distinctive pair of peaks of characteristic relative proportion. Moreover, the NICI mass spectra of the geometrical isomers were significantly different, providing further evidence to validate the identity of any endogenous HNE recovered. The method was used to identify and quantify HNE in platelets, monocytes, plasma and oxidised low-density lipoprotein.     

组蛋白翻译后修饰无标定量方法可靠性的比较

组蛋白翻译后修饰是一种表观遗传学修饰,参与调控细胞的新陈代谢等重要生理过程。蛋白质组学发展迅速,使监控组蛋白翻译后修饰的动态变化成为可能。目前主要有3种无标定量方法（谱图计数法、峰面积积分法和信号强度法）,但何种定量方法更可靠尚未见系统性的详细报道。在稳定同位素标记细胞培养技术（SILAC）基础上,对去乙酰化酶抑制剂（SAHA）调控细胞乙酰化修饰水平的定量数据进行对比,比较3种无标定量方法对组蛋白翻译后修饰进行的定量分析,利用定量结果的标准差（SD）评估定量的可靠性,最终发现基于峰面积积分法定量的结果可靠性最高。该研究对难以进行同位素标记实验的样本分析,尤其对临床样本、大样本的组蛋白修饰谱分析具有重要参考意义。     

Label-free quantification of Tacrolimus in biological samples by atomic force microscopy

In the present paper we describe an atomic force microscopy (AFM)-based method for the quantitative analysis of FK506 (Tacrolimus) in whole blood (WB) samples. Current reference methods used to quantify this immunosuppressive drug are based on mass spectrometry. In addition, an immunoenzymatic assay (ELISA) has been developed and is widely used in clinic, even though it shows a small but consistent overestimation of the actual drug concentration when compared with the mass spectrometry method. The AFM biosensor presented herein utilises the endogen drug receptor, FKBP12, to quantify Tacrolimus levels. The biosensor was first assayed to detect the free drug in solution, and subsequently used for the detection of Tacrolimus in blood samples. The sensor was suitable to generate a dose–response curve in the full range of clinical drug monitoring. A comparison with the clinically tested ELISA assay is also reported.     

Newborn screening of phenylketonuria using direct analysis in real time (DART) mass spectrometry

Phenylketonuria (PKU) is commonly included in the newborn screening panel of most countries, with various techniques being used for quantification of l-phenylalanine (Phe). To diagnose PKU as early as possible in newborn screening, a rapid and simple method of analysis was developed. Using direct analysis in real time (DART) ionization coupled with triple-quadrupole tandem mass spectrometry (TQ-MS/MS) and with use of a 12 DIP-it tip scanner autosampler in positive ion mode, we analyzed dried blood spot (DBS) samples from PKU newborns. The concentration of Phe was determined using multiple reaction monitoring mode with the nondeuterated internal standard N,N-dimethylphenylalanine. The results of the analysis of DBS samples from newborns indicated that the DART-TQ-MS/MS method is fast, accurate, and reproducible. The results prove that this assay as a newborn screen for PKU can be performed in 18 s per sample for the quantification of Phe in DBS samples. DART-TQ-MS/MS analysis of the Phe concentration in DBS samples allowed us to screen newborns for PKU. This innovative protocol is rapid and can be effectively applied on a routine basis to analyze a large number of samples in PKU newborn screening and PKU patient monitoring.

气相色谱-三重四极杆串联质谱稳定同位素稀释法测定被动吸烟儿童尿液中的可丁宁

建立了被动吸烟儿童尿液中可丁宁的气相色谱-三重四极杆串联质谱（GC-MS/MS）稳定同位素稀释测定方法。尿液经过三氯甲烷提取、净化,采用气相色谱-三重四极杆串联质谱多反应监测（MRM）模式测定,以可丁宁-d₃稳定同位素为内标,定量测定和确证被动吸烟儿童尿液中的可丁宁;在0.1~10 μg/L可丁宁质量浓度范围内方法的线性关系良好,相关系数r>0.998;空白尿液中添加可丁宁0.1、1.0和10 μg/L,回收率为79.2%~112.8%,相对标准偏差在2.1%~5.8%之间;方法定量限达到0.1 μg/L。该方法准确、灵敏、快速,适用于家庭被动吸烟儿童尿液中可丁宁的测定。     

凝胶渗透色谱用聚丙烯酸标样的制备

以二氧六环为良溶剂,石油醚为沉淀剂,通过等温沉淀分级,制得8种不同分子量的聚丙烯酸标样。 采用渐近校正法测定了它们的峰值分子量和分子量分布,建立了GPC校正曲线,并将其用于聚丙烯酸试样的测定。 结果表明,由渐进校正法测得的粘均分子量与粘度法测得的分子量之间的平均相对误差为7%,由渐进校正法测得的数均分子量与端基滴定法测得分子量之间的平均相对误差为9%。 将上述标样用于实际聚丙烯酸试样的测定,测得试样的粘均分子量与粘度法测定结果之间平均相对误差小于10%,优于以聚乙二醇为标样测定的平均相对误差（30%左右）。 本标样可用于具有与聚丙烯酸相似结构的阴离子聚电解质分子量分布的测定。     

气相色谱-串联质谱法快速筛查测定中药材中144种农药残留

利用气相色谱-串联质谱(GC-MS/MS)检测技术,采用QuEChERS法作为样品前处理方法,建立了能应用于11种中药材中144种农药残留的检测方法。探究了样品前处理过程中提取溶剂、缓冲盐体系、净化剂组成和用量对样品提取、净化等方面的影响,最终确定了用乙腈提取,甲苯复溶,以混合净化剂净化,过有机膜后经GC-MS/MS测定,外标法定量。144种农药在10~2000 μg/kg之间线性关系良好,相关系数(r²)>0.983;除乙酰甲胺磷、灭虫威、西玛津、克菌丹、异狄氏剂、异菌脲外,其余农药的定量限(LOQ)均低于20 μg/kg;在20、50、200 μg/kg的添加水平下,除乙酰甲胺磷、艾氏剂和双甲脒回收率偏低外,其余141种农药的平均回收率在74.3%~111.8%之间,相对标准偏差(RSD)为0.5%~14.6%。与已有的标准方法对比,此方法不仅检测结果一致,而且高效、快速,准确性好,灵敏度高,适用于中药材中144种农药残留的快速筛查与定量分析。     

Developing On-Site Trace Level Speciation of Lead,Cadmium and Zinc by Stripping Chronopotentiometry (SCP): Fast Screening and Quantification of Total Metal Concentrations

Electrochemical stripping techniques are interesting candidates for carrying out onsite speciation of environmentally relevant trace metals due to the existing low-cost portable instrumentation available and the low detection limits that can be achieved. In this work, we describe the initial analytical technique method development by quantifying the total metal concentrations using Stripping Chronopotentiometry (SCP). Carbon paste screen-printed electrodes were modified with thin films of mercury and used to quantify sub-nanomolar concentrations of lead and cadmium and sub-micromolar concentrations of zinc in river water. Low detection limits of 0.06 nM for Pb(II) and 0.04 nM for Cd(II) were obtained by the standard addition method using a SCP deposition time of 180 s. The SCP results obtained for Pb(II) and Cd(II) agreed with those of inductively coupled plasma mass spectrometry (ICP-MS). The coupling of SCP with screen-printed electrodes opens up excellent potential for the development of onsite speciation of trace metals. Due to the low analysis throughput obtained for the standard addition method, we also propose a new, more rapid screening Cd(II) internal standard methodology to significantly increase the number of samples that can be analyzed per day.     

Sensitive liquid chromatography/mass spectrometry assay for the quantification of azithromycin in human plasma

A simple, rapid, sensitive and specific liquid chromatography/electrospray ionization mass spectrometry method was developed and validated to quantify azithromycin in human plasma, using erythromycin as the internal standard (IS). A simple sample preparation method of protein precipitation with methanol was employed. Methanol, acetonitrile and water (12:30:58, v/v/v) were used as the isocratic mobile phase, with 0.1% formic acid and 0.1% ammonium acetate in water. Selected ion monitoring was specific for azithromycin and erythromycin. The assay was linear over the concentration range 4.69-600 ng/mL. The correlation coefficients for the calibration curves ranged from 0.9994 to 0.9998. The intra- and inter-day precisions, calculated from quality control samples, were less than 8.24%. The method was employed in a pharmacokinetic study after oral administration of 500 mg azithromycin dispersible tablet to 20 healthy volunteers.     

柱前衍生化-气相色谱-质谱法定量测定食品中丙烯酰胺的含量

建立了气相色谱-质谱(GC-MS)定量测定食品中丙烯酰胺含量的分析方法。选择13C3-丙烯酰胺作为内标物。通过超纯水提取食品中的丙烯酰胺,经正己烷脱脂两次后,在酸性条件下选用溴化钾/溴酸钾为衍生剂进行衍生化反应,再采用乙酸乙酯进行液-液萃取两次,最后用三乙胺将丙烯酰胺转化为更稳定的产物2-溴丙烯酰胺,利用质谱检测器在选择离子扫描模式下测定2-溴丙烯酰胺。该方法在0.05～2.00 mg/kg范围具有良好的线性(r2=0.9995);检出限和定量限分别达到3 μg/kg和7 μg/kg;回收率范围为62.7%～65.5%。通过与前期建立的液相色谱-串联质谱(HPLC-MS/MS)方法进行对比,该法在薯片和面包样品中丙烯酰胺的检测结果略偏高,是一种可以用于常见食品中丙烯酰胺含量测定的分析方法。