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.     

Simple ion chromatographic method for the determination of chlormequat residues in pears

Current methods for quantitative determination of chlormequat residues in food crops are characterized by rather low recoveries and the need for derivatization (in case of gas chromatography, GC), or by high capital investment (in case of liquid chromatography–mass spectrometry, LC–MS). We propose a cation-exchange chromatography method for the analysis of chlormequat in pears. The method is based on extraction of the target compound with 40 mM HCl, followed by centrifugation and filtration. The filtrate is directly injected into an ion chromatograph equipped with a commercially available cation-exchange column and a suppressed conductivity detection system. While the limit of detection (LOD) (0.5 mg/kg) may not be small enough to allow dietary analysis, the method meets all validation requirements and is an alternative for the existing GC and LC–MS methods in quality control.     

Challenges in implementing clinical liquid chromatography‐tandem mass spectrometry methods ‐ seeing the light at the end of the tunnel

The use of liquid chromatography–tandem mass spectrometry (LC–MS/MS) in the clinical setting is a relatively new application. One of the significant barriers hampering the transition of LC–MS/MS from the research lab into a clinical setting is the uncertainty of how to successfully develop and validate a method that meets guidelines for clinical applications. Here, we have taken this seemingly overwhelming process and broken it into five general stages for consideration: assessing the clinical validity of a new LC–MS/MS assay, determination of feasibility, assay development, assay validation and post‐implementation monitoring. Although various publications are available and serve as resources for determining development processes and acceptability criteria for specific LC–MS/MS assays, many of them are general recommendations or are specific to research applications that may not translate either practically or clinically. In this perspective special feature article, a resource is compiled that describes key differences between LC–MS/MS methods for research use versus clinical use. In addition, the challenges facing the expanding role of this technique in the clinical setting are discussed, including instrumentation/automation challenges, potential regulation of laboratory developed tests by the US Food and Drug Administration and standardization and harmonization of MS methods through the use of traceable materials and availability of guidance documents. Copyright © 2013 John Wiley & Sons, Ltd.     

Isolation and characterization of methoxylated flavones in the flowers of Primula veris by liquid chromatography and mass spectrometry

Characterization of six flavones, which were named substances G1, G2, G3, G4, G5 and G6 according to their R_F values in normal-phase thin-layer chromatography, is reported. The pure flavones were purified after maceration with methanol by normal-phase solid-phase extraction, normal-phase medium-pressure liquid chromatography, normal-phase preparative thin-layer chromatography and preparative reversed-phase high-performance liquid chromatography (RP-HPLC). The collected fractions of several isolation steps were analyzed by normal-phase (NP) and RP-HPLC. Detection and identification of the substances G was accomplished by UV detection at 213–216 nm, diode array UV detection, or fluorescence detection (λ_ex=330 nm; λ_em=440 nm). The molecular mass, the elementary composition, and the structure of the six components was determined by electron-impact high-resolution mass spectrometry (EI-HRMS). Substance G4 was identified as 3′,4′,5′-trimethoxyflavone. The substances G1–G6 were shown to be mono-, di- tri- and pentamethoxyflavones. HPLC–electrospray ionization tandem mass spectrometry (ESI-MS–MS) of the flavones was carried out employing a 150×2 mm I.D. column packed with a 3 μm/100 Å octadecylsilica stationary phase and a mobile phase comprising 1.0% acetic acid in water–acetonitrile (50:50). Comparative RP-HPLC–ESI-MS of the raw methanol extract and the isolated substances G1–G6 proved that the isolated compounds were pure and were not artifacts. Finally, RP-HPLC–ESI-MS–MS was used to identify substances G1–G6 in phytopharmaceutical drugs.     

Modern developments in gas chromatography-mass spectrometry-based environmental analysis

Gas chromatography coupled with mass spectrometry (GC–MS) continues to play an important role in the identification and quantification of organic contaminants in environmental samples. GC–MS is one of the most attractive and powerful techniques for routine analysis of some ubiquitous organic pollutants due to its good sensitivity and high selectivity and versatility. This paper presents an overview of recent developments and applications of the GC–MS technique in relation to the analysis in environmental samples of known persistent pollutants and some emerging contaminants. The use of different mass analysers such as linear quadrupole, quadrupole ion-trap, double-focusing sectors and time-of-flight analysers is examined. The advantages and limitations of GC–MS methods for selected applications in the field of environmental analysis are discussed. Recent developments in field-portable GC–MS are also examined.     

Determination of diethylhexyl phtalate in water by solid phase microextraction coupled to high performance liquid chromatography

Difficulties detected in the determination of the diethylhexylphthalate (DEHP) at trace levels by gas chromatography–mass spectrometry (GC–MS) using SPME, due to its ubiquitous distribution in the environment has been overcome and a new method for the determination of DEHP in drinking water has been proposed. The method is based on solid phase microextraction (SPME) coupled to high-performance liquid chromatography (HPLC). Detection was carried out spectrophotometrically. Calibration graph was linear in the range 10–110 μg/L with a regression coefficient of r² = 0.998 and a detection limit of 0.6 μg/L. The relative standard deviation was 5 and 2% (n = 4) for chromatographic areas and retention times, respectively. The usefulness of the SPME–HPLC technique was confirmed.     

Mass spectrometric confirmation criterion for product-ion spectra generated in flow-injection analysis. Environmental application

The suitability of a confirmation criterion recently recommended in the Netherlands for gas chromatography with mass spectrometric detection (GC–MS), was evaluated for flow-injection analysis (FIA) with atmospheric pressure chemical ionisation MS–MS detection. The main feature of the criterion is that the relative ion abundances of the four diagnostic ions are taken into account. That is, for lower-intensity peaks, relative standard deviations may be higher; this is an advantage with chemical ionisation MS procedures. A series of triazines and their degradation products were used as test compounds. Tap and surface water samples spiked at 0.33 μg/l were analysed by means of a selected reaction monitoring MS–MS procedure. For all analytes but hydroxysimazine (3 transitions), 4–9 transitions could be selected which invariably met the demands of the criterion. Some of the transitions used originate from the ³⁷Cl isotopic mass of the parent compounds which provides additional structural information. Data for twenty surface water samples analysed by means of FIA–MS–MS as well as GC–MS and liquid chromatography with diode array UV and MS–MS detection gave essentially the same results over the 0.1–1.0 μg/l range. In two samples desethylatrazine was reported by FIA–MS–MS whereas this compound was not detected by GC–MS. For a first test, this is a promising result.     

Quantitation of anabolic hormones and their metabolites in bovine serum and urine by liquid chromatography-tandem mass spectrometry

A specific and sensitive method based on tandem mass spectrometry with on-line high-performance liquid chromatography using atmospheric pressure chemical ionisation (LC–APCI-MS–MS) for the quantitation of anabolic hormone residues (17β-19-nortestosterone, 17β-testosterone and progesterone) and their major metabolites (17-19-nortestosterone and 17-testosterone) in bovine serum and urine is reported. [²H₂]17β-Testosterone was used as internal standard. The analytes were extracted from urine (following enzymatic hydrolysis) and serum samples by liquid–liquid extraction and purified by C₁₈ solid-phase extraction. Ionisation was performed in a heated nebulizer interface operating in the positive ion mode, where only the protonated molecule, [M+H]⁺, was generated for each analyte. This served as precursor ion for collision-induced dissociation and two diagnostic product ions for each analyte were identified for the unambiguous hormone confirmation by selected reaction monitoring LC–MS–MS. The overall inter-day precision (relative standard deviation) ranged from 6.37 to 2.10% and from 6.25 to 2.01%, for the bovine serum and urine samples, respectively, while the inter-day accuracy (relative error) ranged from −5.90 to −3.18% and from −6.40 to −2.97%, for the bovine serum and urine samples, respectively. The limit of quantitation of the method was 0.1 ng/ml for all the hormones in bovine serum and urine. On account of its high sensitivity and specificity the method has been successfully used to confirm illegal hormone administration for regulatory purposes.     

Analysis of acrylamide in different foodstuffs using liquid chromatography–tandem mass spectrometry and gas chromatography–tandem mass spectrometry

Acrylamide levels over a wide range of different food products were analysed using both liquid chromatography–tandem mass spectrometry (HPLC–MS–MS) and gas chromatography–tandem mass spectrometry (GC–MS–MS). Two different sample preparation methods for HPLC–MS–MS analysis were developed and optimised with respect to a high sample throughput on the one hand, and a robust and reliable analysis of difficult matrices on the other hand. The first method is applicable to various foods like potato chips, French fries, cereals, bread, and roasted coffee, allowing the analysis of up to 60 samples per technician and day. The second preparation method is not as simple and fast but enables analysis of difficult matrices like cacao, soluble coffee, molasses, or malt. In addition, this method produces extracts which are also well suited for GC–MS–MS analysis. GC–MS–MS has proven to be a sensitive and selective method offering two transitions for acrylamide even at low levels up to 1 μg kg⁻¹. For the respective methods the repeatability (n=10), given as coefficient of variation, ranged from 3% (acrylamide content of 550 μg kg⁻¹) to 12% (acrylamide content of 8 μg kg⁻¹) depending on the food matrix. The repeatability (n=3) for different food samples spiked with acrylamide (5–1500 μg kg⁻¹) ranged from 1 to 20% depending on the spiking level and the food matrix. The limit of quantification (referred to a signal-to-noise ratio of 9:1) was 30 μg kg⁻¹ for HPLC–MS–MS and 5 μg kg⁻¹ for GC–MS–MS. It could be demonstrated that measurement uncertainties were not only a result of analytical variability but also of inhomogeneity and stability of the acrylamide in food.     

Liquid chromatography-inductively coupled plasma mass spectrometry

It is known that while many elements are considered essential to human health, many others can be toxic. However, because the intake, accumulation, transport, storage and interaction of these different metals and metalloids in nature is strongly influenced by their specific elemental form, complete characterization of the element is essential when assessing its benefits and/or risk. Consequently, interest has grown rapidly in determining oxidation state, chemical ligand association, and complex forms of a many different elements. Elemental speciation, or the analyses that lead to determining the distribution of an element’s particular chemical species in a sample, typically involves the coupling of a separation technique and an element specific detector. A large number of methods have been developed which utilize a multitude of different separation mechanisms and detection instruments. Yet, because of its versatility, robustness, sensitivity and multi-elemental capabilities, the coupling of liquid chromatography to inductively coupled plasma mass spectrometry (LC–ICP–MS) has become one of the most popular techniques for elemental speciation studies. This review focuses on the basic principles of LC–ICP–MS, its historical development and the many ways in which this technique can be applied. Different liquid chromatography separations are discussed as well as the factors that must be considered when coupling each to ICP–MS. Recent applications of LC–ICP–MS to the speciation of environmental, biological and clinical samples are also presented.     

高效液相色谱-电感耦合等离子体质谱联用技术用于环境中元素形态分析的最新进展

综述了高效液相色谱-电感耦合等离子体质谱(HPLC-ICP-MS)联用技术在环境科学中的应用情况.参考过去十年内的国内外文献,从方法学的角度论述了HPLC联用ICP-MS技术的发展,重点探讨了该技术在环境领域,尤其是在元素形态分析(包括生物学,生物无机化学和生物医学)中的应用.     

Development and validation of high-performance liquid chromatography-tandem mass spectrometry assay for 6-(3-benzoyl-ureido)-hexanoic acid hydroxyamide, a novel HDAC inhibitor, in mouse plasma for pharmacokinetic studies

A liquid chromatography/tandem mass spectrometric method for the quantification of 6-(3-benzoyl-ureido)-hexanoic acid hydroxyamide (EX-2), a novel histone deacetylase (HDAC) inhibitor, in mouse plasma was developed to support in-house pharmacokinetic (PK) studies in the lead optimization stage. In order to determine the PK parameters for EX-2 in comparison to other HDAC inhibitors such as suberoylanilide hydroxamic acid (SAHA), PXD-101 and LBH-589, which are currently in different stages of clinical trials, research-grade bio-analytical method validations were carried out for EX-2 and these reference HDAC inhibitors, which were synthesized by in-house medicinal chemists. The components of validation consisted of specificity, extraction efficiency, signal-response of calibration standards, lower limit of quantification, autosampler stability and accuracy and precision of quality control samples. The validated LC/MS/MS methods were accurate and precise. The calibration curve ranged from 1 to 1600 ng/mL for all the analytes. The methods developed were used to quantify EX-2 and other HDAC inhibitors in mouse plasma obtained from pharmacokinetic studies. The results suggest that EX-2 has better PK parameters compared with the reference drugs and is a promising drug development candidate.     

Quantitative analysis of cytokinins in plants by liquid chromatography–single-quadrupole mass spectrometry

A sensitive method for the quantitative analysis of all natural isoprenoid cytokinins in plant material by electrospray single-quadrupole mass spectrometry is presented. A baseline chromatographic separation of 20 non-derivatised naturally occurring cytokinins has been developed. Precise analyses of O-glucoside and ribonucleotide fractions were also performed by the high-performance liquid chromatography–mass spectrometry (HPLC–MS) but run separately from the basic cytokinin metabolites. Using post-column splitting, the flux from narrow-bore (2.1 mm i.d.) reversed-phase liquid chromatography column was simultaneously introduced into the diode array and mass detector. Optimal conditions, including final flow rate, desolvation temperature, desolvation gas flow, capillary and cone voltage for effective ionisation in the electrospray ion source were found. When low cone voltage (20 V) was applied, all studied cytokinins were determined in aqueous methanol as dominant quasi-molecular ions of [M+H]⁺ with limits of detection ranging between 10 and 50 fmol. For routine analysis a linearity range between 25 (75) fmol and 100 pmol was obtained. Developed liquid chromatography–mass spectrometry (LC–MS) method in selective ion monitoring mode was employed to quantify cytokinin species in tobacco BY-2 suspension culture and poplar leaves (Populus×canadensis Moench, cv Robusta).

Purified plant cell (BY-2) and plant tissue (poplar leaves) extracts were obtained by using two different ion-exchange chromatography steps, in combination with immunoaffinity purification using a broad-spectrum monoclonal anti-cytokinin antibody. The antibody strongly recognises the presence of N⁶-substituent on purine skeleton and thus does not bind adenine and related compounds. The presence of authentic cytokinins in the extracts quantified by LC–MS was further verified by enzyme-linked immunosorbent assays (ELISAs) with prior LC preparation. The combination of liquid chromatography–single-quadrupole mass spectrometry with immunoaffinity chromatography offers an efficient and elegant method for detection and quantification of cytokinin metabolites.     

Indirect analysis of urea herbicides from environmental water using solid-phase microextraction

We described here a solid-phase microextraction procedure used to extract six urea pesticides — chlorsulfuron, fluometuron, isoproturon, linuron, metobromuron and monuron — from environmental samples. Two polydimethylsiloxanes and a polyacrylate fiber (PA) are compared. The extraction time, pH control, addition of NaCl to the water and the influence of organic matter such as humic acid on extraction efficiency were examined to achieve a sensitive method. Determination was carried out by gas chromatography with nitrogen–phosphorus detection. The proposed method requires the extraction of 2 ml of sample (pH 4, 14.3%, w/v, NaCl) for 60 min with the PA fiber. The limits of detection range from 0.04 for linuron to 0.1 μg/l for fluometuron and monuron and the relative standard deviations at the 1 μg/l level are between 15% and 9%. The apparent fiber–water distribution constants (K_fw) calculated in the proposed conditions were in the order of 10³. Phenylurea herbicides were indirectly determined in the form of their derived anilines and chlorsulfuron in the form of an aminotriazine as confirmed by gas chromatography–mass spectrometry. Natural waters were utilized to validate the final procedure. However, a unequivocal identification in unknown environmental samples should be done by LC–MS. The presence of dissolved organic matter such as humic acid produces losses during the extraction step. Adding sodium chloride to the sample compensates for this effect.     

A simultaneous TG–DTA study of the thermal decomposition of 2-hydroxybenzoic acid, 2-carboxyphenyl ester (salsalate)

Simultaneous thermogravimetry–differential thermal analysis (TG–DTA) and gas and liquid chromatography with mass spectrometry detection have been used to study the kinetics and decomposition of 2-hydroxybenzoic acid, 2-carboxyphenyl ester, commercially known as salsalate. Samples of salsalate were heated in the TG–DTA apparatus in an inert atmosphere (100 ml min⁻¹ nitrogen) in the temperature range 30–500 °C. The data indicated that the decomposition of salsalate is a two-stage process. The first decomposition stage (150–250 °C) had a best fit with second-order kinetics with E_a=191–198 kJ/mol. The second decomposition stage (300–400 °C) is described as a zero-order process with E_a=72–80 kJ/mol. The products of the decomposition were investigated in two ways:

(a)Salsalate was heated in a gas chromatograph at various isothermal temperatures in the range 150–280 °C, and the exit gas stream analyzed by mass spectrometry (GC–MS). This approach suggested that salsalate decomposes with the formation of salicylic acid, phenol, phenyl salicylate, and cyclic oligomers of salicylic acid di- and tri-salicylides.

(b)One gram samples of salsalate were heated in a vessel under nitrogen to 150 °C, and the residues were analyzed by liquid chromatography–mass spectrometry (LC–MS). The major compound detected was a linear tetrameric salicylate ester.

     

PTV-LV-GC/MS/MS as screening and complementary method to HRMS for the monitoring of dioxin levels in food and feed

Recent developments in trapping efficiency inside ion trap mass spectrometer permitted to lower instrument detection limit (IDL). An IDL of 200 fg μl⁻¹ injected with a signal-to-noise ratio of 5:1 for tetrachlorodibenzo-p-dioxin (TCDD) was obtained by gas chromatography coupled to a quadrupole ion storage mass spectrometer in tandem mode (GC/MS/MS). Coupling large volume programmable temperature vaporizer (PTV-LV) injection to GC/MS/MS provides an alternative and complementary method to classical splitless-GC injection connected to high-resolution mass spectrometry (splitless-GC/HRMS) method for dioxin monitoring in food and feed.

An injection volume of 10 μl was found to be the best compromise between the sensitivity requirements and the robustness required for a high throughput method. PTV-LV-GC/MS/MS and Splitless-GC/HRMS were compared by performing analysis on five different matrices such as beef fat, yolk eggs, milk powder, animal feed and serum samples covering a concentration range of two orders of magnitude (i.e. 0.2–25 ng WHO-TEQ kg⁻¹). An analysis of variance (ANOVA) was carried out. Fisher tests pointed out that the method effect for all the 2,3,7,8 congeners was not significant, indicating that the null hypothesis (H₀: μ₁=μ₂=…=μ_n) was not rejected. Moreover, the interaction effects between methods and matrices were not significant for most of the 2,3,7,8 congeners. However, three congeners (2,3,7,8-TCDF; 1,2,3,4,7,8-HxCDD and 1,2,3,4,6,7,8-HpCDD) were characterized by P-values lower than the significance level (=0.05). In toxic equivalence (TEQ), the study showed that no significant bias was observed between the two methods. Consequently, PTV-LV-GC/MS/MS is an attractive technique and can be used as a cost effective complementary method to HRMS for dioxin levels monitoring in food and feed.     

Evaluation of triclosan and biphenylol in marine sediments and urban wastewaters by pressurized liquid extraction and solid phase extraction followed by gas chromatography mass spectrometry and liquid chromatography mass spectrometry

Analytical methods, based on GC–MS and LC–MS, for the determination of traces of 2,4,4′-trichloro-2′-hydroxydiphenyl ether (triclosan) and biphenylol in urban wastewater and marine sediments were developed. These methods involve the use of diverse analytical techniques, such as solid phase extraction (SPE) and pressurized liquid extraction for sample preparation, and GC–negative chemical ionization MS and LC–electrospray ionization (ESI) MS–MS for identification and quantification. The recoveries of triclosan and biphenylol were 84 and 80% in wastewater and 100 and 73% in sediments, respectively. Detection limits obtained were in the range of ppb and ppt. To prove their applicability to real samples and as part of a more extensive monitoring program, the developed methods were applied to the analysis of wastewater samples, coming from an urban wastewater treatment plant (UWWTP), and of marine sediment samples collected at the outflow of two UWWTPs to the sea. Results obtained reveal the presence of triclosan in all the samples at concentrations that ranged from 0.8 to 37.8 μg/l in wastewater and from 0.27 to 130.7 μg/kg in sediments. These preliminary data reinforce the interest for further research on this topic.     

A rapid ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometric method for the qualitative and quantitative analysis of the constituents of the flower of Trollius ledibouri Reichb

A rapid ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometric (UPLC–ESI-MS/MS) method was developed for the qualitative and quantitative determination of the constituents of the flower of Trollius ledibouri Reichb. The analysis was performed on an AcQuity UPLC™ BEH C₁₈ column using gradient elution with a mobile phase of 0.1% acetic acid and acetonitrile over 20 min. A tandem quadrupole spectrometer operating in either full scan mode or in MS/MS mode for multiple reaction monitoring (MRM) was used for the qualitative and quantitative analysis of the constituents, respectively. According to the mass spectrometric fragmentation mechanism and UPLC–ESI-MS/MS data, the chemical structures of 15 constituents of the flower of T. ledibouri Reichb. were identified on-line without time-consuming isolation and four of them, 2″-O-β-l-galactopyranosylorientin, 2″-O-β-arabinopyranosylorientin, orientin and vitexin, were quantified. The limits of quantification of these four flavonoids were 540, 321, 515 and 220 μg g⁻¹ plant material, respectively. Four commercial samples from different sources were analyzed. The UPLC–ESI-MS/MS method for analyzing the constituents can be used to evaluate the quality of the flower of T. ledibouri Reichb.     

Analytical procedures for quantification of peptides in pharmaceutical research by liquid chromatography–mass spectrometry

Peptide quantification by liquid chromatography–mass spectrometry (LC–MS) combines the high resolving power of reversed-phase (RP) chromatography with the excellent selectivity and sensitivity of mass spectrometric detection. On the basis of comprehensive practical experience in the analysis of small molecules, pharmaceutical research is developing technologies for analysis of a growing number of peptidic drug candidates. This article is a detailed review of procedures based on LC–MS techniques for quantitative determination of peptides. With the focus on pharmaceutical applications several technologies for sample preparation, various aspects of peptide chromatography, important characteristics of ESI–MS, selectivity of MS-detection modes, the large variability of internal standards, and modern instrumentation are discussed. The demand for reliable, robust, sensitive, and accurate methods is discussed using numerous examples from the literature, complemented by experiments and results from our laboratory.