LC-MS/MS signal suppression effects in the analysis of pesticides in complex environmental matrices

The application of LC separation and mobile phase additives in addressing LC-MS/MS matrix signal suppression effects for the analysis of pesticides in a complex environmental matrix was investigated. It was shown that signal suppression is most significant for analytes eluting early in the LC-MS analysis. Introduction of different buffers (e.g. ammonium formate, ammonium hydroxide, formic acid) into the LC mobile phase was effective in improving signal correlation between the matrix and standard samples. The signal improvement is dependent on buffer concentration as well as LC separation of the matrix components. The application of LC separation alone was not effective in addressing suppression effects when characterizing complex matrix samples. Overloading of the LC column by matrix components was found to significantly contribute to analyte-matrix co-elution and suppression of signal. This signal suppression effect can be efficiently compensated by 2D LC (LC-LC) separation techniques. The effectiveness of buffers and LC separation in improving signal correlation between standard and matrix samples is discussed.     

Effect of liquid chromatography separation of complex matrices on liquid chromatography-tandem mass spectrometry signal suppression

The effect of liquid chromatography separation on liquid chromatography-tandem mass spectrometry (LC-MS-MS) signal response for the characterization of low-molecular-mass compounds in a complex matrix was investigated. Matrix induced signal suppression appears throughout the entire LC-MS-MS analysis of wheat forage extract, with greatest suppression occurring at early retention times. Experimental results show that co-elution of matrix components and analytes from the LC column may be most strongly attributed to column overloading rather than similar analyte and matrix retention behavior. As a result, two-dimensional (LC-LC) separation can be a highly effective approach to address signal suppression effects for the quantitative LC-MS-MS analysis of complex matrix samples.     

Quantitative LC-ESI-MS Analysis for Pesticides in a Complex Environmental Matrix Using External and Internal Standards

Abstract

Quantitative liquid chromatography electrospray mass spectrometry (LC-MS) analysis for pesticides in a complex environmental matrix using external and internal standard calibration was investigated. Various approaches to introducing different internal standard compounds to address quantitative errors associated with signal suppression were also examined. The study involved the analysis of pesticides in wheat hay matrix samples using three kinds of internal standard compounds: deuterium labeled (D₃), carbon-13 (singly labeled), and structural analogs (derivatives) of the target analytes. Introduction of the internal standard by volumetric addition and direct post-column infusion were also studied and compared.

Isotopically labeled internal standards (i.e. D₃- ¹³C-) were found to be effective in correcting quantitative errors associated with signal suppression. The application of singly labeled ¹³C compounds may result in nonlinear calibration due to mass interference with the target analyte species. The interference may be compensated by using quadratic curve-fitting or subtraction of the interfering component. Although ineffective as volumetric internal standards, structural analogs can be effective in compensating for signal suppression when introduced into the LC effluent by continuous post-column infusion. Furthermore, the post-column introduction method allows the application of a single internal standard compound for the quantification of each analyte in a multi-component mixture.

The use of internal standards can be effectively incorporated into residue analysis development methods for pesticides in environmental matrices. High accuracy and reproducibility can be achieved while improving method efficiency by reducing the need for comprehensive sample clean-up.     

Effect of the mobile phase composition on the separation and detection of intact proteins by reversed-phase liquid chromatography-electrospray mass spectrometry

Various buffers (ammonium acetate, ammonium formate, and ammonium hydrogencarbonate), acids (formic acid, acetic acid, heptafluorobutyric acid, and trifluoroacetic acid), and bases (ammonium hydroxide and morpholine) covering the range from 2 to 11.5 have been investigated for their performance in the separation of proteins by reversed-phase liquid chromatography (RPLC) and in their detection by electrospray mass spectrometry (ESI-MS). These additives were first tested for the detection of standard proteins by ESI-MS by flow-injection analysis (FIA). Those additives yielding the highest signals were employed for the separation of standard proteins by using three different reversed-phase columns: two C18 columns (4.6 mm I.D. and 2.1 mm I.D.) and one perfusion column (2 mm I.D.). The sensitivity of the LC-MS system was evaluated with the column giving the best results and with those LC eluents enabling the LC separation of the proteins and also yielding the highest MS signals. For that purpose, calibration curves were compared for both LC-MS and FIA-MS. Formic acid was the additive yielding the highest responses in FIA-MS and trifluoroacetic acid (TFA) gave the best separation and recovery of the proteins. However, problems related to poor recovery of the proteins in the column when formic acid was used and the significant signal suppression observed in MS when TFA was employed, made neither of them suitable for the sensitive detection of the proteins in LC-MS.     

液相色谱-串联质谱法检测食品中的多种易滥用着色剂

建立了硬糖、果酱、液态奶、果汁中酸性红52、红色2G、喹啉黄、专利蓝、酸性红26、柠檬黄、靛蓝、胭脂红、诱惑红、日落黄、亮蓝、苋菜红等12种易滥用着色剂残留量的液相色谱-串联质谱分析方法。样品用水溶液稀释提取,经聚酰胺固相萃取柱净化后,在Agilent XDB-C18色谱柱分离,以20 mmol/L乙酸铵溶液和乙腈为流动相进行梯度洗脱。质谱采用电喷雾负离子(ESI~)模式电离,多反应监测(MRM)模式检测,基质匹配标准溶液外标法定量。易滥用着色剂在0.5～50 mg/L范围内呈线性相关,相关系数(r)均大于0.99,其定量限(信噪比大于10)为0.5 mg/kg,检出限(信噪比大于3)为0.1 mg/kg。各种基质样品在0.5、5和50 mg/kg添加水平时,易滥用着色剂的回收率范围为62.6%～115.3%,相对标准偏差(RSDs)为2.6%～26.3%,可以满足食品中易滥用着色剂含量的检测需要。     

采用柱后抑制器技术降低流动相中三氟乙酸对质谱信号抑制作用的研究

研究柱后抑制器(CSRS)技术有效降低流动相中三氟乙酸(TFA)对质谱信号的抑制.采用双三元液相系统,左泵以反相模式分离细胞色素C酶解肽段(流动相含0.1%TFA,流速0.25 m L/min),柱后选择CSRS作为抑制器.同时右泵提供碳酸氢铵(浓度为0.05 mol/L,流速1.00 m L/min)作为再生液.碳酸氢铵和三氟乙酸在抑制器CSRS中通过阴离子交换膜进行离子交换,降低流动相中TFA离子抑制效应,提高肽段在质谱上的响应(S/N提高1~16倍).采用柱后抑制器技术对硫酸依替米星主成分及杂质进行定性分析(0.2 mol/L TFA),流动相经过抑制器后由强酸性变成中性,实现样品在LC和MS之间无缝连接分析.     

Investigation of the effects of 24 bio-matrices on the LC-MS/MS analysis of morinidazole

This study compares and evaluates the effect of various matrices on liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) analysis. Permanent post-column infusion (PCI) was used to quantify matrix effects. In this way, the suppressed or enhanced signal of the target material resulting from different co-eluting matrix components could be assessed. Twenty-four biological samples from in vivo and in vitro experiments were selected for this study. In addition, 7 sample components were further analyzed after sample preparation by protein precipitation. Multiple regression analysis was used to investigate the collinear relationship between matrix effects and co-eluted components at different time intervals. We found that salt was the dominant factor which impacted changes in signal detection. In order to eliminate it, we used ammonium formate as a modifier of the mobile phase which resulted in charge-state redistribution profiles so that a homogeneous matrix formed. By employing pulse gradient chromatography in the presence of 5 mM ammonium formate, favorable improvements of enhanced signal intensity and reduced matrix effects were obtained. These experiments also indicated the feasibility of using analogue IS during bio-analysis which contributed to an overall faster assay that would be suitable for drug discovery and development purposes.     

Characteristics and origins of common chemical noise ions in negative ESI LC-MS

Ionic chemical background noise in LC-MS has been one of the major problems encountered in trace analysis. In this study, the typical negative background ions in ESI LC-MS are investigated exemplarily. It was carried out using tandem mass spectrometry to study the products and precursors of the major background ions to examine their structures and structure relationship. Various typical LC eluents with different compositions and additives such as ammonium formate/formic acid and ammonium acetate/acetic acid have been studied. Several types of negative noise ions are concluded, which include the cluster chemical background ions only from mobile phase components and additives. Furthermore, there are also abundant clusters resulting from the solvation of some typical individual contaminants (e.g. additives and degradation products from tubing, impurities in the mobile phase, etc.), accompanied by some minor contribution from contaminants. The elemental composition of some selected ions was confirmed using the FT-ICR accurate mass measurement. This work provides us insight into information about the structures and types of common negative background ions and will help to understand their formation and origins. More importantly, it will guide us to prevent chemical noise interference in practice and also contribute to develop methods for noise reduction based on selective ion-molecule reactions.     

Micelle-mediated extraction for concentrating hydrophobic organic compounds

An extraction method based on polymer-induced phase separation of aqueous micellar solutions of octyl-beta-D-thioglucoside (OTG) was assessed for concentrating hydrophobic analytes. Various hydrophobic compounds such as polycyclicaromatic hydrocarbons, alkylbenzenes, alkylphenols, chlorobenzenes, chlorophenols, phthalic esters, pesticides, and steroid hormones could be efficiently concentrated into a small volume of surfactant-rich phase, while hydrophilic matrix components remained in the bulk aqueous phase. The surfactant-rich phase containing concentrated OTG could be directly introduced into the hygro-organic mobile phase of high-performance liquid chromatography with ultra-violet photometric detection. The application of this method greatly enhanced the signal intensity in the chromatogram while reducing the interference of matrix components.     

Determination of Antiviral Drugs and Their Metabolites Using Micro-Solid Phase Extraction and UHPLC-MS/MS in Reversed-Phase and Hydrophilic Interaction Chromatography Modes

Two new ultra-high performance liquid chromatography (UHPLC) methods for analyzing 21 selected antivirals and their metabolites were optimized, including sample preparation step, LC separation conditions, and tandem mass spectrometry detection. Micro-solid phase extraction in pipette tips was used to extract antivirals from the biological material of Hanks balanced salt medium of pH 7.4 and 6.5. These media were used in experiments to evaluate the membrane transport of antiviral drugs. Challenging diversity of physicochemical properties was overcome using combined sorbent composed of C₁₈ and ion exchange moiety, which finally allowed to cover the whole range of tested antivirals. For separation, reversed-phase (RP) chromatography and hydrophilic interaction liquid chromatography (HILIC), were optimized using extensive screening of stationary and mobile phase combinations. Optimized RP-UHPLC separation was carried out using BEH Shield RP18 stationary phase and gradient elution with 25 mmol/L formic acid in acetonitrile and in water. HILIC separation was accomplished with a Cortecs HILIC column and gradient elution with 25 mmol/L ammonium formate pH 3 and acetonitrile. Tandem mass spectrometry (MS/MS) conditions were optimized in both chromatographic modes, but obtained results revealed only a little difference in parameters of capillary voltage and cone voltage. While RP-UHPLC-MS/MS exhibited superior separation selectivity, HILIC-UHPLC-MS/MS has shown substantially higher sensitivity of two orders of magnitude for many compounds. Method validation results indicated that HILIC mode was more suitable for multianalyte methods. Despite better separation selectivity achieved in RP-UHPLC-MS/MS, the matrix effects were noticed while using both chromatographic modes leading to signal enhancement in RP and signal suppression in HILIC.     

Multidimensional on-line solid-phase extraction (SPE) using restricted access materials (RAM) in combination with molecular imprinted polymers (MIP)

A novel, multidimensional SPE sample-processing platform for complex fluids, which relies on the combination of small LC columns packed with restricted access materials (RAM) and molecular imprinted polymers (MIP) is described. It is called the Six-S ProcEdure (Six-SPE). Six-SPE involves a size-selective sample-separation step followed by a solvent-switch. Six-SPE efficiently removes interfering matrix components of complex aqueous samples and creates optimal conditions for selective recognition, i.e. binding of the imprinted target analyte(s). A Six-SPE analysis cycle consists of four distinct steps: 1. separation of a given sample (e.g. plasma, urine, saliva, milk, etc.) by adsorptive extraction (e.g. reversed-phase partitioning) of low molecular weight components on to the stationary phase of a RAM column and simultaneous size-exclusion, i.e. quantitative disposal of macromolecular matrix constituents to waste; 2. desorption and transfer of the extract from the RAM column on to a series-connected MIP column using a pure organic mobile phase (e.g. acetonitrile) [solvent switch]; 3. molecular recognition, i.e. selective binding of the target analyte(s) by a tailor-made MIP column; and 4. desorption and transfer of the analyte fraction on to a series-connected separation (e.g. HPLC) and/or detection system (e.g. UV, FD, MS). As a first application we coupled the Six-SPE platform to a conventional HPLC system for on-line analysis of the analgesic drug Tramadol in human plasma using LiChrospher ADS RP-18 as a RAM precolumn for the fractionation step in the first and second chromatographic dimension and a Tramadol imprinted polymer for the molecular recognition step, i.e. third chromatographic dimension.     

Determination of drug enantiomers in biological samples by coupled column liquid chromatography and liquid chromatography-mass spectrometry

Liquid chromatography-mass spectrometry (LC-MS) and coupled column chromatography can be used to overcome problems likely to occur in direct separation and determination of drug enantiomers in biological samples. This is exemplified here with the direct separation and determination of terbutaline in human plasma at the nmol/l level. A beta-cyclodextrin column with an aqueous mobile phase was used for chiral separation. For coupled column chromatography, the concentration of each enantiomer was calculated from the enantiomeric area ratio and the racemate concentration. A deuterium-labelled internal standard was used in the LC-MS experiments.     

'LC-electrolyte effects' improve the bioanalytical performance of liquid chromatography/tandem mass spectrometric assays in supporting pharmacokinetic study for drug discovery

The development of rapid and sensitive bioanalytical methods in a short time frame with acceptable levels of precision and accuracy is imperative for successful drug discovery. We previously reported that the use of a mobile phase containing an extremely low concentration of ammonium formate or formic acid increased analyte electrospray ionization (ESI) response and controlled against matrix effects. We designated these favorable effects 'LC-electrolyte effects'. In order to support rapid pharmacokinetic (PK) studies for drug discovery, we applied LC-electrolyte effects to the development of generic procedures that can be used to quickly generate reliable PK data for compound candidates. We herein demonstrate our approach using four model tested compounds (Compd-A, -B, -C, and -D). The analytical methods involve generic protein precipitation for sample clean-up, followed by application of fast liquid chromatographic (LC) gradients and the subsequent use of electrospray ionization tandem mass spectrometry (ESI-MS/MS) for individual measurement of the tested compounds in 20-microL plasma samples. Good linearity over the concentration range of 1.6 or 8-25000 ng/mL (r(2) > 0.99), precision (RSD, 0.45-13.1%), and accuracy (91-112%) were achieved through the use of a low dose of formic acid (0.4 mM or 0.015 per thousand) in the methanol/water-based LC mobile phase. The analytical method was quite sensitive, providing a lower limit of quantification of 1.6 pg on-column except for Compd-C (8 pg), and showed negligible ion suppression caused by matrix components. Finally, the assay suitability was demonstrated in simulated discovery PK studies of the tested compounds with i.v./p.o. dosing of rats. This new assay approach has been adopted with good results in our laboratory for many recent discovery PK studies.     

A study of ion suppression effects in electrospray ionization from mobile phase additives and solid-phase extracts

Since the wide adoption of liquid chromatography/tandem mass spectrometry (LC/MS/MS), the ion suppression/enhancement phenomenon is the latest barrier to high-throughput analysis. This consequence of a nonoptimized analytical method can lead to adverse effects during quantitation (i.e. poor accuracy and precision). Previous papers have reported that ion suppression is a direct result of endogenous material present in biological samples. However, in the case of a solid-phase liquid chromatography/tandem mass spectrometry (SPE/LC/MS/MS) system, the measured result is the combination of several operating conditions and parameters. Little has been done to effectively monitor and/or choose optimized conditions for the complete sequence of extraction, clean up, separation and analysis. This paper describes a simple setup for quantification of ion suppression/enhancement. Several mobile phase additives, ion-pairing agents and SPE extracts were measured and compared against a standard reference. The results demonstrated that a clean up of plasma extracts based on ion exchange leads to minimal ion suppression/enhancement for the compounds that were investigated.     

Using calibration approaches to compensate for remaining matrix effects in quantitative liquid chromatography/electrospray ionization multistage mass spectrometric analysis of phytoestrogens in aqueous environmental samples

Signal suppression is a common problem in quantitative liquid chromatography/electrospray ionization multistage mass spectrometric (LC/ESI-MS(n)) analysis in environment samples, especially in highly loaded wastewater samples with highly complex matrix. Optimization of sample preparation and improvement of chromatographic separation are prerequisite to improve reproducibility and selectivity. Matrix components are reduced if not eliminated by optimization of sample preparation steps. However, extensive sample preparation may be time-consuming and risk the significant loss of some trace analytes. The best way to further compensate matrix effects is the use of an internal standard for each analyte. However, in a multi-component analysis, finding appropriate internal standards for every analyte is often difficult. In this present study, a more practical alternative option was sought. Matrix effects were assessed using the post-extraction addition method. By comparison of three different calibration approaches, it was found that matrix-matched calibration combined with one internal standard provides a satisfactory method for compensating for any residual matrix effects on all the analytes. Validating experiments on different sewage treatment plant (STP) influent samples analyzing for a range of phytoestrogens showed that this calibration method provided satisfactory results with concentration ratio 96.1-105.7% compared to those by standard addition.     

A simple high pH liquid chromatography-tandem mass spectrometry method for basic compounds: application to ephedrines in doping control analysis

Solvent systems for use with LC-MS often result in a compromise between chromatographic performance and mass spectrometric detection, exemplified here by a LC-MS/MS method development for the analysis of ephedrines in doping control. Ephedrines, frequently found in therapeutic and nutritional preparations, are among the most commonly administered doping agents in competitive sport. Improved separation of these hydrophilic, basic compounds, some of which are diastereoisomers, is achieved in reversed-phase LC by the use of a high pH mobile phase in order to suppress analyte ionisation, and thus alter their polarity, resulting in reduced peak tailing and enhanced retention. However, when coupled to an ESI-MS detector, this eluent composition generated a non-linear and poorly reproducible signal. APCI yielded greater stability and reproducibility and is here presented as an ion source for the analysis of basic compounds under conditions that suppress their ionisation. Errors as large as 49.3% were observed with ESI, compared with 15.4% generated using APCI, for pseudoephedrine over the calibration range (25-400 μg/mL) in urine with a simple dilution and injection of samples. These data highlight the importance of suitable MS conditions for stable performance, necessary for accurate quantification, without undue compromise to the LC separation.     

Simultaneous quantification of multiple licorice flavonoids in rat plasma

Flavonoids are important naturally occurring polyphenols with antioxidant properties. In this study, we report the development of a liquid chromatography tandem mass spectrometry (LC-MS/MS)-based method capable of simultaneously quantifying multiple active licorice flavonoids (including liquiritin apioside, liquiritin, liquiritigenin, isoliquiritin apioside, isoliquiritin, and isoliquiritigenin) in plasma. Electrospray ionization was used to efficiently generate precursor deprotonated molecules of all the analytes and the [M-H]- ions were used to produce characteristic product ions for MS/MS analysis. We found that inclusion of a very low concentration of HCOONH4 (0.01 per thousand) in the LC mobile phase dramatically improved the detection limit for the tested flavonoids and decreased the interference by matrix effects, which have been referred to as "LC-electrolyte effects." Liquid-liquid extraction with ethyl acetate was effective for isolation of all the analytes and resulted in the lowest matrix effects of several tested sample cleanup methods. This bioanalytical method showed good linearity between 0.32 ng/mL and 1 microg/mL analyte in 50-microL plasma samples. The accuracy and precision at different analyte concentrations varied from 85 to 110% and from 0.8 to 8.8%, respectively. Finally, we demonstrated the applicability of this method in a pilot pharmacokinetic study of rats receiving an oral dose of Xiaochaihu-tang, an important Chinese herbal remedy for chronic hepatitis. The use of a low concentration of HCOONH4 in the LC mobile phase could be used to improve LC-mass spectroscopy- or LC-MS/MS-based methods.     

Identification and Recovery of an Asymmetric Calix[4]arene Tetranitrile Derivative using Liquid Chromatography and Mass Spectrometry

A simple analytical liquid chromatography mass spectrometry (LC-MS) method and associated instrumentation has been adapted for use by the organic chemist to yield milligram quantities of target compound from a reaction mixture. Calix[4]arene 3 was identified as representing 51% of total peak area of a reaction mixture containing no less than 10 components, using LC-MS. This peak corresponded to a mass of 878.8, equivalent to a complex of 3 and an ammonium cation. Molecular models further rationalize this observation by showing that the asymmetric binding cavity of 3 is suitable for binding tetrahedral guests such as the ammonium ion. By scaling up the LC method, using analytical instrumentation, 55?mg of 98% pure 3 was isolated with a recovery yield of 90% in 1?h. The current method represents a powerful and easily adapted tool for monitoring a challenging synthesis that combines identification, efficient separation and partial characterization for reaction mixture components using readily available instrumentation and methods.     

二维液相色谱-质谱联用分离中药复方葛根芩连汤中的有效成分

建立了二维液相色谱-质谱联用方法分离中药复方葛根芩连汤的成分。以CN柱作第一维色谱柱,水和甲醇梯度洗脱分离;以ODS柱作第二维色谱柱,20 mmol/L乙酸铵缓冲液和乙腈梯度洗脱分离;质谱检测采用电喷雾电离/大气压化学电离（ESI/APCI）复合离子源,正负离子扫描。实验结果表明搭建的二维液相色谱的峰容量显著高于一维色谱,分离效率得到了明显的提高。以第一维色谱的第3个流分为例,对其二维分离进行仔细分析,发现质谱比紫外光谱检测到的组分多,质谱中采用负离子模式比正离子模式检测到的组分多。表明搭建的二维液相色谱-质谱分离平台分离效果好,提高了液相色谱的峰容量和分离效率。该方法操作简便,可作为中药等复杂体系分离分析的有效手段。     

Selective and Sensitive LC-MS Determination of Avenanthramides in Oats

A new liquid chromatographic – mass spectrometric (LC-MS) method suitable for the determination of avenanthramides in oats has been developed. The avenanthramides were detected using positive ion electrospray with selective ion monitoring of protonated ions [M+H]⁺. The separation of avenanthramides was achieved on Genesis C₁₈ column by using acetonitrile – aqueous formic acid as mobile phase and gradient elution. The effects of buffer nature and concentration on separation, peak shape and intensity of mass spectrometric signal were investigated. The detection limits ranged between 10 and 30 ng mL^?1 for standard solutions when using 1 μL injection. The developed LC-MS method was found to be superior over commonly used liquid chromatographic methods with UV detection in terms of selectivity and sensitivity. The applicability of the method to real samples (oat groats) was demonstrated.