Liquid chromatography/fast-atom-bombardement mass spectrometry of nonionic detergents

Summary Microcolumn liquid chromatography (LC) combined with fast-atom-bombardment mass spectrometry (FABMS) was applied to the analysis of nonionic detergents. The porous silver filter attached to the top of the capillary fused-silica tubing worked as the interface for coupling of micro LC and FABMS.     

Separation and identification of ceramides in the human stratum corneum by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry and electrospray multiple-stage mass spectrometry profiling

Summary A sensitive, selective, and rapid method is described for analysis of ceramides in the human stratum coracum by direct coupling of HPLC with an electrospray ion-trap mass spectrometry. Nonaqueous reversed-phase chromatography stabilizes the electrospray ionization, resulting in sensitivity that enables direct measurement of skin lipid extracts with no special sample preparation. Assignment of individual signals to the corresponding ceramide species is based on interpretation of the fragment spectra from MS-MS experiments. This enables much finer differentiation between ceramdies than that achievable by thin-layer chromatography. Summary A sensitive, selective, and rapid method is described for analysis of ceramides in the human stratum corneum by direct coupling of HPLC with an electrospray ion-trap mass spectrometry. Nonaqueous reversed-phase chromatography stabilizes the electrospray ionization, resulting in sensitivity that enables direct measurement of skin lipid extracts with no special sample preparation. Assignment of individual signals to the corresponding ceramide species is based on interpretation of the fragment spectra from MS-MS experiments. This enables much finer differentiation between ceramides than that achievable by thin-layer chromatography.     

Liquid chromatography/electrospray tandem mass spectrometry of terpenoid lactones in Ginkgo biloba

Ginkgo biloba (ginkgo) is one of most frequently used botanical dietary supplements. The bioactive constituents include the terpenoid lactones consisting of bilobalide and the ginkgolides A, B, C and J. A new assay based on high-performance liquid chromatography/electrospray tandem mass spectrometry (LC/MS/MS) was developed for the measurement of the terpenoid lactones in ginkgo products such as leaf powder and extracts. Initially, the MS/MS fragmentation pathways of ginkgolides were investigated to identify abundant fragment ions that might be useful for the sensitive and selective detection of ginkgolides and bilobalide during LC/MS/MS. Then, sample preparation and clean-up procedures were streamlined to maximize throughput by taking advantage of the selectivity of LC/MS/MS detection. Analyte recoveries exceeded 90%, the intra-assay and inter-assay relative standard deviations were <5%, the relative error was <8% and the limits of detection and quantification were 3.6-120 and 11-350 fmol, depending on the analyte that was injected on to the LC column. Therefore, this LC/MS/MS assay facilitated the rapid quantitative analysis of ginkgolides A, B, C and J and bilobalide in ginkgo dietary supplements with excellent recovery, reproducibity, accuracy and sensitivity.     

In vivo and in vitro metabolism of aspirin eugenol ester in dog by liquid chromatography tandem mass spectrometry

Aspirin eugenol ester (AEE) is a promising drug candidate for treatment of inflammation, pain and fever and prevention of cardiovascular diseases with fewer side effects than its precursor, aspirin. Investigation into its metabolic process in target animal species will help to illustrate its mechanism of action and to establish its residual mark compound to formulate its dosage. Six beagle dogs were orally given a dose of 20 mg kg^?1 of AEE and one dog was used to prepare blank liver microsomes. Their liver microsomes were prepared for in vitro study and their plasma and urine were collected for in vivo metabolic analysis using liquid chromatography tandem mass spectrometry. In this study we identified 10 metabolites, M1, M2, M3, M4, M5 in phase I and M6, M7, M8, M9, M10 in phase II. Based on the metabolites of AEE, the pathways of AEE metabolism in dog were demonstrated. Copyright © 2014 John Wiley & Sons, Ltd.     

Dicationic ion-pairing of phosphoric acid diesters post-liquid chromatography and subsequent determination by electrospray positive ionization-tandem mass spectrometry

Several organophosphate triesters are widely used as flame retardants and can be metabolized to dibutyl (DBP), diphenyl (DPhP), di(2-ethylhexyl) (DEHP) and di(1,3-dichloro-2-propyl) (or bis(1,3-dichloro-2-propyl); DDCPP) phosphoric acid, respectively. A highly sensitive liquid chromatography-electrospray ionization(+)-triple quadrupole mass spectrometry (LC-ESI(+)-QQQ-MS/MS) based analysis method was presently developed. In this method the target compounds were separated with a C(18)-based reversed phase LC column, and decamethonium hydroxide (dicatonic reagent) was introduced post-LC to form ion-pairs, which were subsequently detected by ESI(+). For the phosphate acid diester ion-pairs, the mass spectra showed the most abundant ion to be [(CH(3))(2)N(CH(2))(10)N(CH(3))(3)](+), with lesser abundances of [[M-H](-)[(CH(3))(3)N(CH(2))(9)CH(2)](2+)](+) and [CH(2)CH(CH(2))(8)N(CH(3))(3)](+). For DDCPP, the fragment ions of [[Cl](-)[(CH(3))(3)N(CH(2))(10)N(CH(3))(3)](2+)](+) and [[Cl](-)[(CH(3))(3)N(CH(2))(9)CH(2)](2+)](+) could also be observed. The limits of quantitation (LOQs) by LC-ESI(+)-MS/MS (based on multiple reaction monitoring) were 0.14, 0.03, 0.14 and 0.02 ng/mL for DPhP, DBP, DDCPP and DEHP, respectively. The response was highly linearly correlated (r>0.995) with concentration over the range of the LOD to 1000 ng/mL. The matrix effect on ESI+ was negligible for the samples in experiment of in vitro metabolism using rat liver microsomes.     

Simultaneous determination of a new phosphodiesterase-5 inhibitor DA-8159 and its active metabolite in human plasma by high performance liquid chromatography with tandem mass spectrometry

Summary An LC-MS-MS method for the simultaneous determination of DA-8159 and its active metabolite, DA-8164 in human plasma was developed. DA-8159, DA-8164 and the internal standard, sildenafil were extracted from human plasma with dichloromethane at basic pH. A reversephase HPLC separation was performed on Luna phenylhexyl column with the mixture of acetonitrile-ammonium formate (10 mM, pH 6.0) (60:40,v/v) as mobile phase. The detection of analytes was performed using an electrospray ionization tandem mass spectrometry in the multiple reaction monitoring mode. The method showed a satisfactory sensitivity (lower limits of quantification, 2.0 ng mL¹), precision, accuracy, recovery and selectivity. The successful determination of DA-8159 and DA-8164 in the plasma of a volunteer who ingested a single dose of 100 mg DA-8159 confirms that the present method can be used for plasma analysis for clinical trial.     

Profiling ABA metabolites in Nicotiana tabacum L. leaves by ultra-performance liquid chromatography-electrospray tandem mass spectrometry

We have developed a simple method for extracting and purifying (+)-abscisic acid (ABA) and eight ABA metabolites - phaseic acid (PA), dihydrophaseic acid (DPA), neophaseic acid (neoPA), ABA-glucose ester (ABAGE), 7′-hydroxy-ABA (7′-OH-ABA), 9′-hydroxy-ABA (9′-OH-ABA), ABAaldehyde, and ABAalcohol - before analysis by a novel technique for these substances, ultra-performance liquid chromatography-electrospray ionisation tandem mass spectrometry (UPLC-ESI-MS/MS). The procedure includes addition of deuterium-labelled standards, extraction with methanol-water-acetic acid (10:89:1, v/v), simple purification by Oasis^® HLB cartridges, rapid chromatographic separation by UPLC, and sensitive, accurate quantification by MS/MS in multiple reaction monitoring modes. The detection limits of the technique ranged between 0.1 and 1 pmol for ABAGE and ABA acids in negative ion mode, and 0.01-0.50 pmol for ABAGE, ABAaldehyde, ABAalcohol and the methylated acids in positive ion mode. The fast liquid chromatographic separation and analysis of ABA and its eight measured derivatives by UPLC-ESI-MS/MS provide rapid, accurate and robust quantification of most of the substances, and the low detection limits allow small amounts of tissue (1-5 mg) to be used in quantitative analysis. To demonstrate the potential of the technique, we isolated ABA and its metabolites from control and water-stressed tobacco leaf tissues then analysed them by UPLC-ESI-MS/MS. Only ABA, PA, DPA, neoPA, and ABAGE were detected in the samples. PA was the most abundant analyte (ca. 1000 pmol/g f.w.) in both the control and water-stressed tissues, followed by ABAGE and DPA, which were both present at levels ca. 5-fold lower. ABA levels were at least 100-fold lower than PA concentrations, but they increased following the water stress treatment, while ABAGE, PA, and DPA levels decreased. Overall, the technique offers substantial improvements over previously described methods, enabling the detailed, direct study of diverse ABA metabolites in small amounts of plant tissue.     

Optimization for detection of flunarizine by high performance liquid chromatography/electrospray/mass spectrometry

This paper describes a newly developed high performance liquid chromatography/electrospray/mass spectrometry (HPLC/ES/MS) method for the determination of flunarizine (FZ) in artificial cerebrospinal fluid. The optimization for the detection of FZ in biological fluid by LC/ES/MS was investigated. The effects of solvent composition, the addition of modifier and flow rate on the detection of FZ by ES/MS were examined. The detection limit of this method (0.8 nM) proved to be much better than previously reported methods. Satisfactory accuracy (98.2–106.0%) of this newly developed method was obtained. The application of this method was demonstrated by analyzing FZ in rat microdialysis samples.     

液相色谱-电喷雾串联质谱法测定冬青芽中的脱落酸

建立了测定植物中痕量脱落酸 (ABA) 的液相色谱-电喷雾串联质谱联用分析方法. 植物提取液先采用固相萃取 (SPE) 富集脱落酸并消除基体干扰, 然后以C18柱为固定相, V(甲醇)∶V(2 g/L甲酸水溶液)＝50∶50为流动相分离脱落酸. 电喷雾(ESI)串联质谱采用负离子模式. 选择反应监测 (SRM) 模式用于脱落酸定量, 选择的离子对是263→153, 219. 脱落酸的线性范围为0.005～10 μg/mL, 检测限 (S/N=3) 为0.003 μg/mL, 加标0.02 μg和0.05 μg的回收率分别为98.3% 和103.5%. 该方法用于冬青芽中脱落酸的分析, 结果表明所建立的SPE-LC-MS/MS方法对于分析植物中的痕量脱落酸是有效的.     

Mass spectrometry in the study of anthocyanins and their derivatives: differentiation of Vitis vinifera and hybrid grapes by liquid chromatography/electrospray ionization mass spectrometry and tandem mass spectrometry

A mass spectrometric-based procedure for anthocyanin profiling was set up to distinguish authentic Vitis vinifera from hybrid red grapevine cultivars. 3-O-Monoglucoside and the related acetyl-, p-coumaryl- and caffeoyl-monoglucoside anthocyanins occurred only in Vitis vinifera, whereas 3,5-O-diglucoside and the substituted acetyl-, p-coumaryl-, feruloyl- and caffeoyl-diglucoside anthocyanins were the additional pigments in hybrid grapevines. The procedure was applied expressly to identify red grape cultivars based on the anthocyanin chemo-type determination. In particular, a red grape cultivar, having 3,5-O-diglucoside anthocyanins and a novel class of anthocyanin monoglucosides, such as cyanidin-3-O-, cyanidin-3-O-(6-O-acetyl)- and cyanidin-3-O-(6-O-p-coumaryl)pentoside, was classified as hybrid. A second vine cultivar, characterized exclusively by 3-O-monoglucoside anthocyanins, was included among the Vitis vinifera species. Anthocyanin profiling by mass spectrometry could represent the core of a chemotaxonomic procedure for distinguishing American and European grapevines based on the identification of post-synthetic anthocyanidin modification.     

Combination of liquid chromatography/tandem mass spectrometry and gas chromatography/mass spectrometry for the detection of 21 anabolic steroid residues in bovine urine

For the detection of anabolic steroid residues in bovine urine, a highly sensitive liquid chromatographic/electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method was developed using both positive and negative ionization. For four compounds the ESI mode was not sensitive enough and gas chromatographic/mass spectrometric GC/MS detection was therefore still necessary as a complementary method. The sample clean-up consisted of solid-phase extraction (SPE) on a C(18) column followed by enzymatic hydrolysis and a second solid-phase extraction on a combination of a C(18) and a NH(2) column. After this last SPE clean-up, the eluate was split into two equal aliquots. One aliquot was further purified and after derivatization used for GC/MS analysis. The other aliquot was analyzed with LC/MS/MS in both ESI+ and ESI- modes. The method was validated according to the European Commission Decision 2002/657/EC. Decision limits (CCalpha) were between 0.16 and 1 ng ml(-1) for the compounds detected with the LC/MS/MS method. The developed method is used in routine analysis in our laboratory.     

Column liquid chromatography coupled on-line with mass spectrometry using post-column phosphate suppression

The coupling of column liquid chromatography on-line with mass spectrometry using post-column phosphate suppression is described. A membrane suppressor is used to remove the non-volatile phosphate ions from the mobile phase after the chromatographic separation prior to introduction into the mass spectrometer. Using post-column phosphate suppression by a membrane device phosphate concentrations up to 10 mM phosphate, at a mobile phase flow rate of 0.1 ml/min, could be removed for more than 99% without any decay of the sensitivity of the MS detection. Using a new antidepressant tetracyclic basic compound, ORG 4428, and some structure related compounds as model compounds electron impact as well as chemical ionisation spectra of all compounds could be obtained. The set-up could be used for several weeks running, using a phosphate containing mobile phase, without any loss of performance.     

Ultra-high performance liquid chromatography tandem mass spectrometry for comprehensive analysis of urinary acylcarnitines

We report an enabling mass spectrometric method for the analysis of lipid metabolites in order to define better the lipid metabolome in terms of chemical diversity and generate fragment ion spectra of these metabolites as a potential resource for unknown metabolite identification. This work focuses on the analysis of one important class of lipid metabolites, the acylcarnitines. Current analytical methods have only detected and identified a limited number of these metabolites. The method described herein provides the most comprehensive acylcarnitine profile in urine of healthy individuals up to date. It involves an optimized solid phase extraction technique for selective analyte extraction using cartridges containing both lipophilic and cation-exchange properties. The captured analytes are then subjected to ultra-high performance liquid chromatography (UPLC) separation, followed by tandem mass spectrometry (MS/MS) analysis using information-dependent acquisitions and selected reaction monitoring (SRM). The urine of six healthy individuals was analyzed using this method. A total of 355 acylcarnitines were detected; only 43 of them have been previously reported in the urine of healthy individuals. Detection of this large number of acylcarnitines illustrates the great diversity of the lipid metabolome as well as the usefulness of the method for profiling acylcarnitines. Furthermore, the MS/MS spectra of the 355 acylcarnitines will be uploaded to a public human metabolome database as a mass spectrometric resource for unknown metabolite identification.     

Liquid chromatography-electrospray ionization tandem mass spectrometry and liquid chromatography with fluorescence detection for determination of octylphenol and nonylphenol in municipal wastewater at trace levels

Summary Reversed-phase liquid chromatography with fluorescence detection (RPLC-FD) and with electrospray ionization (ESI) tandem mass spectrometric detection (RPLC-ESI-MS-MS), both coupled with automated solid-phase sample extraction, have been used for trace enrichment and analysis of octylphenol and nonylphenol (NP) in municipal wastewater. Quality data calculated for the methods were detection limits (LOD), quantitation limits, linearity, precision, and accuracy. For LC-ESI-MS-MS theLOD were below 0.08 μg L⁻¹. Unequivocal detection of NP in wastewater samples was achieved by use of LC-ESI-MS and LC-ESI-MS-MS. In particular, application of the LC methods to wastewater samples revealed that the selectivity of LC-MS-MS was more than that of LC-FD for qualitative and quantitative analysis of complex matrices.     

Validation of a method for the analysis of quinolones residues in bovine muscle by liquid chromatography with electrospray ionisation tandem mass spectrometry detection

A liquid chromatography-tandem mass spectrometry method for the determination and confirmation of nine quinolones was optimised and validated according to Commission Decision 2002/657/EC. Analytes were extracted from veal muscle with water and extracts purified with 96-well plates Oasis HLB cartridges. Separation was carried out in a silica-based C₁₈ column (50 mm × 2.1 mm) with mobile phases consisting of water/acetonitrile mixtures containing acetic acid. Linear calibration curves in the ranges 4-400 and 50-800 ng g⁻¹, with correlation coefficients at least 0.995, were obtained for all the analytes. At concentration levels above 10 ng g⁻¹, quantification errors were lower than 10% and repeatability and within-laboratory reproducibility standard deviations below 6% and 10%, respectively. Decision limits and detection capabilities are reported.     

Characterization of peptides by liquid chromatography/electrospray ionization mass spectrometry using silver nitrate as a post-column complexant

Two model peptides, des-Arg1-bradykinin (DAB) and bradykinin (B), were cationized by Ag+ after their separation by reversed-phase liquid chromatography (RPLC) prior to mass spectrometry (MS). Silver nitrate solution was used as a post-column reagent. The RPLC and MS experimental conditions were optimized using flow injection in order to obtain sufficiently abundant silver adducts to permit MS/MS experiments. The use of water-methanol with 0.1% formic acid as mobile phase allowed a good chromatographic separation of the two peptides with a polymeric stationary phase and sufficiently abundant silver-containing adducts, [M + Ag + H]2+ and [M + 2Ag]2+. The gas-phase dissociation of [DAB + Ag + H]2+ and [DAB + 2Ag]2+ led to interpretable mass spectra during the on-line cationization experiment. Most of the ions obtained by dissociating [DAB + Ag + H]2+ and [DAB + 2Ag]2+ species are silver-containing ions but the ions produced depend on the parent. The ions coming from the dissociation of the doubly charged silver adducts [DAB + Ag + H]2+ or [DAB + 2Ag]2+ are of interest compared with those coming from the singly charged silver species or doubly charged protonated species. The fragmentation of the doubly charged silver adducts provides ions over the entire mass range. Although the presence of several prolines in des-Arg1-bradykinin prevents the formation of some expected ions, the observation of triplets [an-H + Ag]+, [bn-H + Ag]+ and [bn + OH + Ag]+ produced by the dissociation of on-line Ag(+)-cationized peptides could contribute to greater success of automatic sequencing of peptides.     

Quantitative liquid chromatography/time-of-flight mass spectrometry

Over the last decade, time-of-flight (TOF) instruments have increasingly been used as quantitation tools. In addition, because of their high resolving power, they can be used for verification of empirical formulas. Historically, TOF instruments have had limited quantitation capabilities because of their narrow dynamic range. However, recent advances have improved these limitations. This review covers the rationale for using TOF for LC detection, and describes the many methods currently in the literature for the quantitation of pharmaceuticals, environmental pollutants, explosives and many phytochemicals.     

Liquid chromatography coupled to tandem mass spectrometry for the analysis of inositol hexaphosphate after solid-phase extraction

Inositol hexaphosphate, a naturally occurring component in cereals and plants, is the main reserve and principal carrier of phosphate. Inositol hexaphosphate is also found in biological fluids such as urine, plasma, or whole blood. Moreover, inositol hexaphosphate is studied for its pharmaceutical applications. Liquid chromatography coupled to mass spectrometry is now the reference method for small analyte determination. However, the specific quantitation of polyanionic molecules in the biological matrix is still challenging.

In this article, a bioanalytical method for the extraction and determination of inositol hexaphosphate in whole blood is described by using solid-phase extraction followed by liquid chromatography–mass spectrometry/mass spectrometry using selected reaction monitoring mode for its specificity.

Using pentylamine in excess, an ion pair is created, enhancing sensitivity by avoiding the presence of many Na adducts on the phosphate functions of inositol hexaphosphate. Moreover, hexafluoroisopropanol was added to stabilize the ion pairs that were created. Then, a specific extraction of inositol hexaphosphate by anion-exchange solid-phase extraction was developed, resulting in an extraction recovery of 89%. The linearity of the method was verified between 0.78 and 100 µg/mL, and both accuracy and precision were greater than 85%. Finally, the endogenous rate of inositol hexaphosphate was measured in the whole blood of mice and was estimated at 2 µg/mL.     

Simultaneous screening of 21 potential adulterants in dietary supplements for the treatment of prostate diseases using ultra-performance liquid chromatography and liquid chromatography-electrospray ionization-tandem mass spectrometry

Reports of the number of adulteration cases using illegal therapeutic substances in dietary supplements have increased. In recent years, various dietary supplements are being distributed that exaggerate the efficacy of treatment for prostate-related diseases. To develop the preemptive monitoring method, we selected 21 prostate-related therapeutic substances and optimized the simultaneous ultra-performance liquid chromatography and liquid chromatography-electrospray ionization-tandem mass spectrometry and pretreatment procedures for various types of matrices including solid, liquid, and soft capsule samples. The methods were validated by determining the specificity, linearity, limit of detection, limit of quantification, method detection limit, method quantitation limit, precision, accuracy, recovery, stability, and matrix effect. The simultaneous methods were validated according to the international guidelines. In addition, using the validated methods, 81 real samples, which were searched and purchased by focusing on promotional phrases, such as prostate and prostatic hyperplasia, were successfully screened. As a result, sildenafil and tadalafil were detected in one seized capsule sample (5.15 and 14.6 mg/g, respectively). Synthetically, our approach could be useful for the determination of illegal therapeutic substances potentially adulterated in various types of dietary supplements.     

Determination of green-tea catechins in cosmetic formulations and in in-vitro skin extracts by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry

Summary A simple, rapid and reproducible method is presented for the analysis of green-tea extracts in different cosmetic formulations and in in-vitro skin extracts. Cosmetically active principal components were used for determination of complex assembled green-tea extracts. The catechins selected were catechin, epicatechin, epigallocatechin, epigallocatechin gallate, and epicatechin gallate, because of their efficacy and their concentrations in green-tea extracts. The determination was performed by high-performance liquid chromatography (HPLC), on a reversed-phase (RP) column, coupled to a single-quadrupole mass spectrometer (MS) via an electrospray ionization (ESI) interface. Detection was performed in the negative selected-ion-monitoring (SIM) mode. A detection limit between 5 and 15 ng g⁻¹ was achieved in methanol-water-ascorbic acid extracts from different emulsions. A routine analytical procedure could be established with good quantitative reliability. During validation, the repeatabilities (relative standard deviation) for catechin standard solutions were found to be 1.1–2.7% (within one day) and 2.2–4.3% (day-to-day). Recoveries from spiked placebos were 98–105%. The method was successfully used to determine the storage stability of green-tea extract in cosmetic formulations and the in-vitro penetration of green-tea extract into the skin.