Liquid chromatography/mass spectrometry in anabolic steroid analysis--optimization and comparison of three ionization techniques: electrospray ionization,atmospheric pressure chemical ionization and atmospheric pressure photoionization

The applicability of liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the detection of the free anabolic steroid fraction in human urine was examined. Electrospray ionization (ESI), atmospheric pressure chemical ionization and atmospheric pressure photoionization methods were optimized regarding eluent composition, ion source parameters and fragmentation. The methods were compared with respect to specificity and detection limit. Although all methods proved suitable, LC/ESI-MS/MS with a methanol-water gradient including 5 mM ammonium acetate and 0.01% acetic acid was found best for the purpose. Multiple reaction monitoring allowed the determination of steroids in urine at low nanogram per milliliter levels. LC/MS/MS exhibited high sensitivity and specificity for the detection of free steroids and may be a suitable technique for screening for the abuse of anabolic steroids in sports.     

Measurement of the malondialdehyde-2'-deoxyguanosine adduct in human urine by immuno-extraction and liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry

The major adduct of malondialdehyde with guanine, M(1)G, was measured in human urine from non-smoking healthy individuals. M1G is a mutagenic DNA lesion and a terminal product of lipid peroxidation in vivo that may be implicated in cancer related to lifestyle and diet. On the basis of a recently developed method for the quantification of M1G as an excreted deoxynucleoside using immuno-extraction purification, chemical NaBH4 reduction and liquid chromatography combined with atmospheric pressure chemical ionization tandem mass spectrometry, we demonstrate that the average 24 h excretion rate of M1G-dR is about 12 +/- 3.8 fmol kg(-1) (n = 5).     

Separation and quantitation of nicergoline and related substances by high-performance liquid chromatography/atmospheric pressure ionization mass spectrometry

Summary This study demonstrated the utility of high-performance liquid chromatography/atmospheric pressure ionization mass spectrometry (HPLC/API-MS) in the investigation of 10-methoxy-1,6-dimethylergoline-8-methanol 5-bromonicotinic acid ester (Nicergoline) and its related substances. The analysis was performed by using an ODS column with ammonium acetate and methanol mixture as the mobile phase. Nicergoline and its related compounds could be characterized by HPLC/API-MS in terms of their molecular weight. The use of multiple ion detection techniques for the quantitation of these compounds was also investigated. The detection limits of nicergoline and its related substances were 5 to 10 ng each at a signal-to-noise ratio of 4. The method was also applied to the study of the decomposition products of nicergoline in simulated gastric and intestinal fluids.     

Comparison of electrospray ionization and atmospheric pressure chemical ionization techniques in the analysis of the main constituents from Rhodiola rosea extracts by liquid chromatography/mass spectrometry

Rhodiola rosea L. (Golden Root) has been used for a long time as an adaptogen in Chinese traditional medicine and is reported to have many pharmacological properties. A liquid chromatographic (LC) method with mass spectrometric (MS) detection based on selected ion monitoring (SIM) was developed for determining salidroside, sachaliside 1, rosin, 4-methoxycinnamyl-O-beta-glucopyranoside, rosarin, rosavin, cinnamyl-(6'-O-beta-xylopyranosyl)-O-beta-glucopyranoside, 4-methoxy-cinnamyl-(6'-O-alpha-arabinopyranosyl)-O-beta-glucopyranoside, rosiridin and benzyl-O-beta-glucopyranoside from the callus and plant extracts in one chromatographic run. Good linearity over the range 0.5-500 ng ml(-1) for salidroside, 2-2000 ng ml(-1) for rosavin and 2-500 ng ml(-1) for benzyl-O-beta-glucopyranoside was observed. The intra-assay accuracy and precision within quantitation ranges varied between -10.0 and +13.2% and between 0.7 and 9.0%, respectively. Optimization of the ionization process was performed with electrospray and atmospheric pressure chemical ionization techniques using four different additive compositions for eluents in the LC/MS scan mode, using both positive and negative ion modes. The best ionization sensitivity for the compounds studied was obtained with electrospray ionization when using pure water without any additives as the aqueous phase.     

液相色谱-大气压化学电离离子阱质谱法测定烟草中的游离茄尼醇

用液相色谱/大气压化学电离离子阱质谱建立了一种分析烟草中游离茄尼醇的方法。烟草样品用甲醇振荡提取30 min,在分析前无需进行其它前处理。在1.8μm快速分离C18色谱短柱上用V(甲醇)∶V(异丙醇)=85∶15等梯度洗脱实现了茄尼醇的快速分离。用不带碰撞能量的二级质谱全扫描选择监测离子m/z 613.6进行定量,检出限为0.4μg/L,RSD为1.1%,两种添加量的回收率分别为97%和99%。方法应用于不同烟草和烟草制品样品的检测分析。     

Analysis of thiamine in dried yeast by high-performance liquid chromatography and high-performance liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry

Summary High-performance liquid chromatography (HPLC) and high-performance liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (HPLC/APCI-MS) have been applied to the analysis of thiamine in dried yeast. Thiamine was extracted from dried yeast with isobutanol containing sodium 1-octanesulfonate as an ion-pairing agent and determined by HPLC on a reversed phase ODS column with UV detection at 254 nm. Response was linear in the range 25–300 μg/g of thiamine in dried yeast with a coefficient of variation in the reproducibility of 8.0%. Thiamine was recovered in good yield (109.2%, n=5). Identification of the thiamine peak was obtained by the mass spectrum using the HPLC/APCI-MS system. The utility of the selected ion monitoring technique using the HPLC/APCI-MS was also investigated. The results obtained by this method are in good agreement with those obtained by the thiochrome method [1].     

Analysis of native amino acids by liquid chromatography/electrospray ionization mass spectrometry: comparative study between two sources and interfaces

The analytical performances of two triple-quadrupole instruments, which differ in their atmospheric-pressure sources, were evaluated for native amino acid analysis. The Applied Biosystems/Sciex API 300 instrument was equipped with a turboIon Spray source and a curtain gas interface while the Waters/Micromass Quattro Ultima instrument was characterized by its Z-spray source. Liquid chromatography/mass spectrometry analysis of native amino acids requires volatile ion-pairing mobile phase additives (mainly perfluorinated carboxylic acids). The effects of the structure and concentration of the ion-pairing reagents as well as the organic modifier percentage on the electrospray response of amino acids were studied in detail. The most favourable chromatographic conditions depend strongly on the mass spectrometer used. Several instrumental parameters were also studied, including spray voltage, transmission lens voltages, temperature of desolvation and auxiliary gas flow rates. The results show substantial qualitative differences depending on the instrument geometry. The quantitative performances of the two triple-quadrupole mass spectrometers were evaluated in terms of limits of detection and quantification. The effects of the matrix on the analyte ionization were also examined, and the long-term stability of the electrospray performance was studied over 12 h using a mobile phase containing the perfluorinated ion-pairing reagents. The study provides information on the robustness of the MS instrument and its detection sensitivity towards native amino acid analysis. It appears that each instrument has its good and bad points since one provides higher sensitivity while another is more robust.     

Analysis of FeII-mediated decomposition of a linoleic acid-derived lipid hydroperoxide by liquid chromatography/mass spectrometry

Intracellular Fe(II), which is up-regulated during oxidative stress and during iron overload, induces the formation of a hydroxyl radical by Fenton chemistry. The hydroxyl radical can convert the prototypic omega-6 polyunsaturated fatty acid, linoleic acid, to 13-hydroperoxy-9,11-(Z,E)-octadecadienoic acid (13-HPODE). Cyclooxygenases can also convert linoleic acid to 13(S)-HPODE during oxidative stress. Subsequent Fe(II)-mediated decomposition to protein- and DNA-reactive bifunctional electrophiles was examined by normal-phase liquid chromatography (LC)/atmospheric pressure chemical ionization (APCI)/mass spectrometry. The potential individual bifunctional electrophiles trans-4,5-epoxy-2(E)-decenal (EDE), cis-EDE, 4-oxo-2(E)-nonenal (ONE) and 4-hydroxy-2(E)-nonenal (HNE) exhibited protonated molecular ions at m/z 169, 169, 155 and 157, respectively. The MH(+) ion at m/z 173 for 4-hydroperoxy-2(E)-nonenal (HPNE) was very weak with an ion corresponding to the loss of OH at m/z 156 as the major ion in the APCI mass spectrum. The bifunctional electrophiles were all separated under normal-phase LC conditions. Interestingly, ions corresponding to ONE and HNE were detected at the same retention time as HPNE, suggesting that it decomposed in the source of the mass spectrometer to ONE and HNE. All five bifunctional electrophiles were formed when 13-HPODE was treated with 50 microM Fe(II). At this concentration of Fe(II), the addition of vitamin C resulted in increased bifunctional electrophile formation. At higher concentrations of Fe(II) (500 microM to 2 mM), no HPNE was detected and there was no additive effect of vitamin C. Additional experiments with synthetic HPNE revealed that it was quantitatively converted to a mixture of ONE and HNE by Fe(II). The HNE is thought to arise from a one-electron reduction of an alkoxy radical derived from HPNE. In contrast, ONE can arise through an alpha-cleavage of the HPNE-derived alkoxy radical or by direct dehydration of HPNE.     

Atmospheric pressure chemical ionization mass spectrometry and in-source fragmentation of lutein esters

Negative-ion atmospheric pressure chemical ionization (APCI) mass spectrometry and in-source collisionally induced dissociation (CID) were employed to obtain structural information of lutein esters from marigold extract. Both molecular ions and structurally significant fragments corresponding to the loss of fatty acids were observed in high abundance in the current study. Six lutein diesters including lauroylmyristoyl-lutein (LML), dimyristoyl-lutein (dML), myristoylpalmitoyl-lutein (MPL), dipalmitoyl-lutein (dPL), palmitoylstearoyl-lutein (PSL) and distearoyl-lutein (dSL) were characterized in a marigold flower extract. Breakdown curves (plots of relative ion abundance vs. internal energy) of three lutein diesters were established by monitoring the relative ion abundance of molecular and fragment ions at different cone voltages during negative-ion APCI-LC/MS analysis.     

Document authentication at molecular levels using desorption atmospheric pressure chemical ionization mass spectrometry imaging

Molecular images of documents were obtained by sequentially scanning the surface of the document using desorption atmospheric pressure chemical ionization mass spectrometry (DAPCI‐MS), which was operated in either a gasless, solvent‐free or methanol vapor‐assisted mode. The decay process of the ink used for handwriting was monitored by following the signal intensities recorded by DAPCI‐MS. Handwritings made using four types of inks on four kinds of paper surfaces were tested. By studying the dynamic decay of the inks, DAPCI‐MS imaging differentiated a 10‐min old from two 4 h old samples. Non‐destructive forensic analysis of forged signatures either handwritten or computer‐assisted was achieved according to the difference of the contour in DAPCI images, which was attributed to the strength personalized by different writers. Distinction of the order of writing/stamping on documents and detection of illegal printings were accomplished with a spatial resolution of about 140 µm. A Matlab® written program was developed to facilitate the visualization of the similarity between signature images obtained by DAPCI‐MS. The experimental results show that DAPCI‐MS imaging provides rich information at the molecular level and thus can be used for the reliable document analysis in forensic applications. © 2013 The Authors. Journal of Mass Spectrometry published by John Wiley & Sons, Ltd.     

Determination of polycyclic aromatic hydrocarbons in fractions in asphalt mixtures using liquid chromatography coupled to mass spectrometry with atmospheric pressure chemical ionization

An analytical method using liquid chromatography coupled to mass spectrometry with atmospheric pressure chemical ionization for the determination of polycyclic aromatic hydrocarbons in asphalt fractions has been developed. The 14 compounds determined, characterized by having two or more condensed aromatic rings, are expected to be present in asphalt and are considered carcinogenic and mutagenic. The parameters of the atmospheric pressure chemical ionization interface were optimized to obtain the highest possible sensitivity for all of the compounds. The limits of detection ranged from 0.5 to 346.5 μg/L and the limits of quantification ranged from 1.7 to 1550 μg/L. The method was validated against a diesel particulate extract standard reference material (NIST SRM 1975), and the obtained concentrations agreed with the certified values. The method was applied to asphalt samples after its fractionation according to ASTM D4124 and the method of Green. The concentrations of the seven polycyclic aromatic hydrocarbons quantified in the sample ranged from 0.86 mg/kg for benzo[ghi]perylene to 98.32 mg/kg for fluorene.     

银离子高效液相色谱-质谱法分析血清中甘油三酯类化合物的组成

针对甘油三酯(TAG)类化合物的复杂性,建立了分析小鼠血清中TAG类化合物的方法。采用经典的氯仿-甲醇溶剂体系对血中的TAG类化合物进行提取。脂质提取物经Varian ChromSpher 5 Lipids柱分离,在0.75 mL/min的流速下以乙腈-正己烷(1:99, v/v)为流动相进行等度洗脱,采用大气压化学电离源正离子模式电离,质谱增强型全扫描、增强型子离子扫描和中性丢失扫描模式检测。根据银离子色谱对双键的保留规律以及质谱所给出的碎片离子信息,对血清中TAG类化合物进行了结构鉴定。结果表明采用该方法可以从小鼠血清中鉴定到66个TAG类化合物以及5个胆固醇酯。该方法简单,重现性好,可通用于其他样品中TAG类化合物的检测。     

Characterization and quantification of triacylglycerols in peanut oil by off‐line comprehensive two‐dimensional liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry

The complexity of natural triacylglycerols (TAGs) in various edible oils is prodigious due to the hundreds of set is of TAG compositions, which makes the identification of TAGs quite difficult. In this investigation, the off‐line 2D system coupling of nonaqueous RP and silver‐ion HPLC with atmospheric pressure chemical ionization MS detection has been applied to the identification and quantification of TAGs in peanut oil. The method was successful in the separation of a high number of TAG solutes, and the TAG structures were evaluated by analyzing their atmospheric pressure chemical ionization mass spectra information. HPLC and MS conditions have been optimized and the fragmentation mechanisms of isomers have been validated. In addition, an internal standard approach has been developed for TAG quantification. Then this system was applied in peanut oil samples and there was a total of 48 TAGs including regioisomers that have been determined and quantified.     

大气压离子化技术/飞行时间质谱联用进展

综述了大气压离子化技术/飞行时间质谱联用技术及其应用的进展     

Systematic screening and characterization of glycosides in tobacco leaves by liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry using neutral loss scan and product ion scan

Glycosides in tobacco leaves are highly important aromatic precursors. It is necessary to reveal glycosides in tobacco leaves to improve tobacco planting and processing. This study describes a method for the systematic screening of glycosides in tobacco leaves by liquid chromatography with tandem mass spectrometry. Although glycosides contain numerous aglycones, the number of glycans is limited. Based on a screening table of glycans designed for neutral loss scan, glycosides with different aglycones were systematically screened out. Then, the MS² fragment spectra of scanned glycosides were further obtained using product ion scan. By comparison with the spectra in online tandem mass spectral databases, reported references, and verification by commercial standards, 64 glycosides were detected, including 39 glycosides linked with monosaccharides, 18 glycosides linked with disaccharides and 7 glycosides linked with trisaccharides. It is noteworthy that glycosides linked with trisaccharides have previously been rarely reported in tobacco. This method appears to be a useful tool for the systematic screening and characterization of glycosides in tobacco and can potentially be applied to other plants.     

Super‐atmospheric pressure chemical ionization mass spectrometry

Super‐atmospheric pressure chemical ionization (APCI) mass spectrometry was performed using a commercial mass spectrometer by pressurizing the ion source with compressed air up to 7 atm. Similar to typical APCI source, reactant ions in the experiment were generated with corona discharge using a needle electrode. Although a higher needle potential was necessary to initiate the corona discharge, discharge current and detected ion signal were stable at all tested pressures. A Roots booster pump with variable pumping speed was installed between the evacuation port of the mass spectrometer and the original rough pumps to maintain a same pressure in the first pumping stage of the mass spectrometer regardless of ion source pressure. Measurement of gaseous methamphetamine and research department explosive showed an increase in ion intensity with the ion source pressure until an optimum pressure at around 4–5 atm. Beyond 5 atm, the ion intensity decreased with further increase of pressure, likely due to greater ion losses inside the ion transport capillary. For benzene, it was found that besides molecular ion and protonated species, ion due to [M + 2H]⁺ which was not so common in APCI, was also observed with high ion abundance under super‐atmospheric pressure condition. Copyright © 2013 John Wiley & Sons, Ltd.     

Atmospheric pressure photoionization-mass spectrometry and atmospheric pressure chemical ionization-mass spectrometry of neurotransmitters

A group of five neurotransmitters with different properties was analyzed using atmospheric pressure photoionization (APPI) and atmospheric pressure chemical ionization-mass spectrometry (APCI-MS). The sensitivity of the techniques for the analytes was tested in six solvents and in positive and negative ion modes. APPI was found to be superior in sensitivity for all the compounds in both positive and negative ion modes. In positive ion mode, water/methanol/formic acid was found to be the best solvent, whereas in negative ion mode, water/methanol/ammonium hydroxide performed best. Detection limits using APPI were between 2.5-250 fmol, depending on the compound. The sensitivity was best for the neurosteroids dehydroepiandrosterone and beta-estradiol, and acetylcholine (LOD 2.5-10 fmol).     

超高效液相色谱-大气压化学电离-串联质谱法测定烘焙咖啡中丙烯酰胺

建立了烘焙咖啡中丙烯酰胺的超高效液相色谱-大气压化学电离-串联质谱（UHPLC-APCI-MS/MS）分析方法。样品经甲醇提取，HLB固相萃取（SPE）小柱净化，Brownlee validated AQ C18色谱柱分离，采用大气压化学电离（APCI）源，正离子扫描和多反应监测（MRM）模式对丙烯酰胺进行检测，内标法定量。结果表明，丙烯酰胺在0.5~100.0 μg/L范围内具有良好的线性关系，相关系数（r²）为0.999，方法检出限为5.0 μg/kg，定量限为10.0 μg/kg。在100.0、200.0和1000.0 μg/kg添加水平下，丙烯酰胺的回收率为94.6%~115.0%，相对标准偏差（RSD）值为2.8%~3.6%（n=6）。本方法采用APCI源作为离子化方式，能有效地减少咖啡基质对丙烯酰胺的基质干扰，前处理简单，灵敏度高，适用于咖啡中丙烯酰胺的日常检测。     

Determination of eight nitrosamines in water at the ng L levels by liquid chromatography coupled to atmospheric pressure chemical ionization tandem mass spectrometry

In this work, we have developed a sensitive method for detection and quantification of eight N-nitrosamines, N-nitrosodimethylamine (NDMA), N-nitrosomorpholine (NMor), N-nitrosomethylethylamine (NMEA), N-nitrosopirrolidine (NPyr), N-nitrosodiethylamine (NDEA), N-nitrosopiperidine (NPip), N-nitroso-n-dipropylamine (NDPA) and N-nitrosodi-n-butylamine (NDBA) in drinking water. The method is based on liquid chromatography coupled to tandem mass spectrometry, using atmospheric pressure chemical ionization (APCI) in positive mode with a triple quadrupole analyzer (QqQ). The simultaneous acquisition of two MS/MS transitions in selected reaction monitoring mode (SRM) for each compound, together with the evaluation of their relative intensity, allowed the simultaneous quantification and reliable identification in water at ppt levels. Empirical formula of the product ions selected was confirmed by UHPLC-(Q)TOF MS accurate mass measurements from reference standards.Prior to LC–MS/MS QqQ analysis, a preconcentration step by off-line SPE using coconut charcoal EPA 521 cartridges (by passing 500 mL of water sample) was necessary to improve the sensitivity and to meet regulation requirements. For accurate quantification, two isotope labelled nitrosamines (NDMA-d₆ and NDPA-d₁₄) were added as surrogate internal standards to the samples.The optimized method was validated at two concentration levels (10 and 100 ng L⁻¹) in drinking water samples, obtaining satisfactory recoveries (between 90 and 120%) and precision (RSD < 20%). Limits of detection were found to be in the range of 1–8 ng L⁻¹. The described methodology has been applied to different types of water samples: chlorinated from drinking water and wastewater treatment plants (DWTP and WWTP, respectively), wastewaters subjected to ozonation and tap waters.     

Differentiation of human kidney stones induced by melamine and uric acid using surface desorption atmospheric pressure chemical ionization mass spectrometry

Clinically obtained human kidney stones of different pathogenesis were dissolved in acetic acid/methanol solutions and then rapidly analyzed by surface desorption atmospheric pressure chemical ionization mass spectrometry (SDAPCI-MS) without any desalination treatment. The mass spectral fingerprints of six groups of kidney stone samples were rapidly recorded in the mass range of m/z 50-400. A set of ten melamine-induced kidney stone samples and nine uric acid derived kidney stone samples were successfully differentiated from other groups by principal component analysis of SDAPCI-MS fingerprints upon positive-ion detection mode. In contrast, the mass spectra recorded using negative-ion detection mode did not give enough information to differentiate those stone samples. The results showed that in addition to the melamine, the chemical compounds enwrapped in the melamine-induced kidney stone samples differed from other kidney stone samples, providing useful hints for studying on the formation mechanisms of melamine-induced kidney stones. This study also provides useful information on establishing a MS-based platform for rapid analysis of the melamine-induced human kidney stones at molecular levels.