Effects of ion-pairing reagents on the electrospray signal suppression of sulphonated dyes and intermediates

High-performance liquid chromatography/mass spectrometry (HPLC/MS) analysis of anionic species such as sulphonic acid dyes and intermediates requires volatile ion-pairing mobile phase additives. Six di- and trialkylammonium acetates were compared with tetraalkylammonium salts and ammonium acetate in the concentration range 0-20 mmol l(-1) as mobile phase additives for HPLC/MS of polysulphonated compounds. The effects of the structure and concentration of the ion-pairing reagents on the electrospray response of mono-, di- and tetrasulphonic aromatic acids and acid dyes were studied in detail. Further, five different mass analysers and instrument geometries were compared. A higher signal decrease is observed with linear geometry instruments in comparison to orthogonal or even Z-spray geometry mass spectrometers. The concentration of mobile phase additives has a significant influence on the abundance ratios of multiply charged ions in the mass spectra of polysulphonated compounds. The competing ions of sulphonic acids may also cause significant signal suppression.     

Ion‐pairing high‐performance liquid chromatography‐mass spectrometry of impurities and reduction products of sulphonated azodyes

An HPLC separation method with triethylammonium acetate mobile phase additive developed for the analysis of impurities in polysulphonated azo dyes provides good separation selectivity and compatibility with electrospray ionisation (ESI) mass spectrometry. The negative‐ion ESI mass spectra containing only peaks of deprotonated molecules [M–H]^– for monosulphonic acids, [M–xH]^x–, and sodiated adducts [M–(x + y)H + yNa]^x– for polysulphonic acids allow easy molecular mass determination of unknown impurities. Based on the knowledge of the molecular masses and of the fragment ions in the MS/MS spectra, probable structures of trace impurities in commercial dye samples are proposed. To assist in the interpretation of the mass spectra of complex polysulphonated azodyes, additional information can be obtained after chemical reduction of azodyes to aromatic amines. The structures of the non‐sulphonated reduction products can be determined by reversed‐phase HPLC/MS with positive‐ion atmospheric pressure chemical ionisation and of the sulphonated products by ion‐pairing HPLC/MS with negative‐ion ESI.     

Separation of isomeric naphthalenesulphonic acids by micro high-performance liquid chromatography with mobile phases containing cyclodextrin

Aromatic sulphonic acids are important dye intermediates and the determination of the individual isomers after their preparation by sulphonation of the parent aromatic hydrocarbon is important for the monitoring of the dye production process. For this purpose, either reversed-phase chromatography with mobile phases containing strong electrolytes as additives or capillary zone electrophoresis with working electrolytes containing cyclodextrins can be used to separate and determine not only individual sulphonation products with various numbers of sulphonic groups, but also various isomeric di- and trisulphonic acids. However, the separation of some isomers using either of the two techniques is not fully satisfactory. In the present work, HPLC with mobile phases containing cyclodextrins was employed to improve previously achieved separations of aromatic sulphonic acids. Because of the high cost of cyclodextrin, microcolumn HPLC with diode-array detection on the columns prepared in laboratory by supercritical fluid packing technique was employed for this purpose. Capillary columns packed with various octadecyl silica gel materials were compared and their stability and efficiency were found suitable for the separation of the compounds tested. The selectivity of separation of some isomers improved significantly with respect to the previous methods. Procedures were designed for separation and analytical control of technological processes producing dye intermediates.     

The role of analogue ions in the ion-pair reversed-phase chromatography of quaternary ammonium compounds

The choice of analogue ion of the mobile phase additive is shown to significantly affect the analysis of quaternary ammonium compounds (QACs) in ion-pair reversed-phase high-performance liquid chromatography. A series of bromide-containing and dodecyl-sulfate-containing mobile phase additives are investigated using two QAC probe analytes. In all instances, the quaternary-ammonium-containing mobile phase additives perform better than the corresponding sodium-containing additives for effective QAC elution. These results indicate that the structure of the analogue ion, not just its formal charge, is important in the reversed-phase ion-pair chromatography of these compounds. The relative elution order of the QAC probe analytes is also influenced by the counter ions of the mobile phase additives, with bromide and dodecyl sulfate offering opposite elution orders.     

The hydrophilic interaction like properties of some reversed phase high performance liquid chromatography columns in the analysis of basic compounds

The hydrophilic interaction chromatography (HILIC) like properties of an ACE cyano (CN) HPLC column was studied for the separation of some basic compounds. Good separation of a test mix of basic compounds was obtained with a mobile phase consisting of acetonitrile/water (95:5) containing 3.25 mM ammonium acetate. The retention times of the basic compounds decreased with increased ionic strength or with increased water content in the mobile phase. When Trishydroxymethyl aminomethane (Tris) (pK(a) 8.0), which is a weaker amine than ammonia (pK(a) 9.3), was used as an additive in the mobile phase retention of the basic compounds was increased. The ACE CN column gave excellent peak shapes for all the basic compounds. The utility of the column for impurity profiling of two basic drugs was tested and some impurities in oxprenolol were characterised by interfacing with Fourier transform mass spectrometry. It was also observed that ACE butyl and ACE phenyl columns retained basic compounds when the columns were eluted with a mobile phase consisting of acetonitrile/water (95:5) containing 3.25 mM ammonium acetate.     

Simultaneous separation of hydrophobic and hydrophilic peptides with a silica hydride stationary phase using aqueous normal phase conditions

The application of a silica hydride modified stationary phase with low organic loading has been investigated as a new type of chromatographic material suitable for the separation and analysis of peptides with electrospray ionization mass spectrometric detection. Retention maps were established to delineate the chromatographic characteristics of a series of peptides with physical properties ranging from strongly hydrophobic to very hydrophilic and encompassing a broad range of pI values (pI 5.5-9.4). The effects of low concentrations of two additives (formic acid and acetic acid) in the mobile phase were also investigated with respect to their contribution to separation selectivity and retention under comparable conditions. Significantly, strong retention of both the hydrophobic and the hydrophilic peptides was observed when high-organic low-aqueous mobile phases were employed, thus providing a new avenue to achieve high resolution peptide separations. For example, simultaneous separation of hydrophobic and hydrophilic peptides was achieved under aqueous normal phase (ANP) chromatographic conditions with linear gradient elution procedures in a single run, whilst further gradient optimization enabled improved peak efficiencies of the more strongly retained hydrophobic and hydrophilic peptides.     

缓冲盐类型和离子对试剂非对离子对强离解酸性化合物离子对反相液相色谱保留行为的影响

在离子对反相液相色谱(IP-RPLC)分析中,溶质保留受对离子(counter ion)的影响比较受人关注,但鲜有研究流动相中缓冲盐类型和离子对试剂中非对离子(non-counter ion)对溶质保留行为的影响。鉴于此,该文以14种磺酸化合物为研究对象,甲醇为有机调节剂,分别考察了3种缓冲盐体系(磷酸二氢铵、氯化铵和乙酸铵)和5种离子对试剂体系(四丁基溴化铵、四丁基磷酸二氢铵、四丁基硫酸氢铵、四丁基硝酸铵和四丁基乙酸铵)下强离解酸性化合物的IP-RPLC保留行为,通过比较不同流动相条件下得到的溶质log k_w(100%水相作流动相时的保留因子)、S(线型溶剂强度模型线性回归得到的常数),以及CHI(色谱疏水指数,log k_w/S),寻找保留行为规律。研究表明,流动相中的缓冲盐类型和离子对试剂非对离子均会影响化合物的log k_w和S值,所有化合物在氯化铵缓冲盐体系下具有最大的log k_w值。相对于无机阴离子,离子对试剂中弱离解性有机阴离子(乙酸根)的存在有利于增加磺酸化合物的S值。通过对比不同条件下的保留行为,推测磺酸化合物的IP-RPLC保留机理中同时存在着离子对模型和动态离子交换模型。与log k_w和S值不同,化合物的CHI值受缓冲盐类型以及离子对试剂非对离子的影响较弱。此外,研究发现化合物的表观正辛醇/水分配系数(log D)与log k_w、S、CHI之间均具有良好的线性相关性。不同缓冲溶液和不同离子对试剂非对离子条件下获得的log k_w和S值存在着一定的差异,而CHI值相对稳定,因此,CHI更适用于IP-RPLC中定量结构-保留行为关系模型的建立。     

Reversed-phase high-performance liquid chromatography of amphoteric beta-lactam antibiotics: effects of columns, ion-pairing reagents and mobile phase pH on their retention times

The separation of five amino beta-lactam antibiotics by reversed-phase high-performance liquid chromatography was studied as an insight into their retention behaviour. These five amphoteric compounds are cephradine, cephalexine, cefaclor, ampicillin and amoxicillin. Both octadecylsilane-bonded silica (C18) columns and phenyl-bonded silica (phenyl) columns were used, with mobile phase pH values between 2.5 and 7.4. In the absence of ion-pairing reagents the retention times for all the five compounds were the shortest at pH 4-6. The phenyl column was found to improve the separation between cephradine and ampicillin at pH values lower than 3, when these two compounds appeared as fused peaks on the C18 on C18 columns, with mobile phases both with and without ion-pairing reagents, were compared. The addition of 0.005 or 0.02 M tetraethylammonium acetate to the mobile phase did not result in significant ion-pair formation, except at pH values higher than 5.5. A strong ion-pairing effect was obtained at pH values higher than 6 with 0.005 or 0.02 M tetrabutylammonium phosphate, and the retention was decreased at pH values lower than 4. On the other hand, 0.005 M heptanesulphonic acid exhibited an ion-pair retention effect at pH values lower than 5. The molecular structures and pK(a) values were used to account for the retention behaviour of these antibiotics in the various mobile phases.     

Screening and confirmation analysis of stimulants, narcotics and beta-adrenergic agents in human urine by hydrophilic interaction liquid chromatography coupled to mass spectrometry

The chromatographic behaviour of 44 polar compounds (23 beta-adrenergic agents, 11 stimulants, 4 narcotics and 6 phenolalkylamines) included in the list of prohibited substances and methods of the World Anti-Doping Agency, has been investigated under hydrophilic interaction liquid chromatography conditions by application of different mobile phase compositions (percentage of the organic solvent, type and amount of mobile phase additive and ionic strength) and column temperatures. Detection of analytes was performed by a triple quadrupole mass spectrometer in positive ionization mode and selected reaction monitoring acquisition mode after liquid/liquid extraction. Data collected using as stationary phase type-B silica materials from different producers, showed that the best chromatographic conditions in terms of peak shape, selectivity and chromatographic retention were obtained using an initial percentage of acetonitrile of 90%, a column temperature of 35 °C, a mobile phase pH of 4.5 and ammonium acetate (5 mM) and acetic acid (0.1%) as mobile phase additives. The selected chromatographic conditions were used to develop screening and confirmation analytical procedures to detect polar compounds in human urine for antidoping purpose. The developed methods were validated in terms of specificity, matrix effect, linearity, precision, accuracy, sensitivity, robustness and repeatability of retention times and relative ion abundances. Such methods offer attractive alternatives and considerable advantages over traditional approaches especially for the analysis of the phenolalkylamines.     

Sequential optimisation of the separation of a complex mixture of plastics additives by HPLC with a quaternary gradient and a dual detection system

Summary The separation of two families of plastics additives (phenolic antioxidants and UV absorbers) has been achieved by high performance liquid chromatography with a quaternary gradient. A methodology of separation based on a sequential optimization is described. After a preliminary study of the effects of solvents on retention of compounds, the mobile phase is first chosen for each class of additives (Irganox and Tinuvin), then for the mixture of all the compounds and the separation is finally optimized. The importance of the column phase ratio is also reported. The use of two detectors, UV absorbance and light-scattering, enables all the compounds to be detected. The performance of the detectors has been compared and the effects of the nebulization temperature on the detection of low molecular mass compounds is reported.     

Optimization of Conditions for the HPLC Determination of Synthetic Dyes in Food

The retention of synthetic food dyes on the sorbent Kromasil C18 was studied under the conditions of reversed-phase and ion-pair high-performance liquid chromatography, depending on the concentrations of the ion-pair reagent (tetrabutylammonium dihydrophosphate) and organic solvent (acetonitrile) in the mobile phase and on the concentration and pH of the phosphate buffer solution. The optimum conditions were selected for the separation of ten dyes in the isocratic elution mode within 25 min. A procedure was developed for the determination of synthetic food dyes in foodstuffs; the detection limit was 10–30 g/L.     

多糖衍生物手性固定相上采用酸性或碱性添加剂的流动相拆分β受体阻滞剂对映体

考察了多糖类手性固定相在含有酸性或碱性添加剂的流动相下高效液相色谱法拆分β受体阻滞剂对映体的效果。色谱条件: 流动相为10%～30%(体积分数,下同)乙醇-正己烷(含0.1%三氟乙酸)和10%～30%乙醇-正己烷(含0.1%三乙胺),流速1.0 mL/min,紫外检测波长254 nm。结果表明,在直链淀粉-三(3,5-二甲基苯基氨基甲酸酯)衍生物手性固定相(Chiralpak AD和Chiralpak IA)上拆分β受体阻滞剂对映体,酸性添加剂的流动相体系与碱性添加剂的流动相体系相比,碱性添加剂的流动相的拆分效果比酸性添加剂的流动相要好。而在纤维素-三(3,5-二甲基苯基氨基甲酸酯)衍生物的手性固定相(Chiralcel OD和Chiralpak IB)上分离β受体阻滞剂,比较酸性添加剂的流动相与碱性添加剂的流动相的拆分效果,发现酸性添加剂的流动相条件下对映体的保留减弱,但对映体的选择性增大,特别是在Chiralcel OD上,酸性添加剂的流动相体系对对映体的选择性非常理想,而且随着流动相中酸性添加剂含量的增加,β受体阻滞剂对映体的分离效果更佳。     

Mass spectrometry based analysis of nucleotides, nucleosides, and nucleobases—application to feed supplements

In this work, accurate MS-based methods for quantitative profiling of nucleotides, nucleosides, and nucleobases in yeast extracts used as additives in animal feedstuff are presented. Reversed-phase chromatography utilizing a stationary phase compatible with 100% aqueous mobile phases resulted in superior analytical figures of merit than HILIC or ion-pair reversed-phase separation. The novel separation method was combined with both molecular and elemental mass spectrometry. By use of RP-LC-MS-MS, excellent limits of detection <1 μmol L(-1) could be obtained for all the compounds investigated. The elemental speciation analysis approach enabled determination of nucleotides by phosphorus detection. Sensitivity of LC-ICP-MS was 1-2 orders of magnitude lower than that of LC-MS-MS. Quantitative analysis of yeast products using complementary MS detection furnished values in good agreement.     

Novel additives for the separation of organic acids by ion-pair chromatography

<正>This paper proposes the use of novel surfactant additives for the separation of organic acids by ion-pair chromatography and studies the influences of surfactants on the chromatographic separation behaviors.Researches have been carried out on both silica gel matrix and polymer supporters in order to compare the two ordinary kinds of stationary phases,and the phenomenon is similar. Separation is based on differences in the stabilities of analyte-additive complexes in solution.Retention times of analytes can be varied over a large range by varying the additive concentration.The results indicate that the additives of proper concentrations can reduce the retention times of different organic acids while the resolution remains the same.The larger the molecular weight is,the greater the shift of the retention time is.This greatly expands the scope of macromolecular polar compounds that can be separated by ion-pair chromatography with the advantages of retention times being greatly reduced.     

Separation of N-nitrosoamino acids by C18 reversed-phase ion-pair high-performance liquid chromatography and compatible detection by electrospray ionization mass spectrometry.

Four non-volatile N-nitrosoamino acids, namely N-nitrososarcosine, N-nitrosoproline, N-nitrosothiazolidine-4-carboxylic acid and N-nitroso-2-methyl-thiazolidine-4-carboxylic acid were separated by C18 reversed-phase ion-pair high-performance liquid chromatography (HPLC) using 1.4 mM C16-cetyltrimethylammonium chloride in methanol-water-acetonitrile (60:35:5, v/v) as the mobile phase. The N-nitrosoamino acids were sensitively detected by negative electrospray ionization mass spectrometry (ESI-MS) in the form of the deprotonated carboxylate anion, [M-H]-. Compatibility problems associated with HPLC separation and ESI-MS detection, such as formation of solvent cluster ions and the effects of co-eluting anions of the ion-pairing reagent, were systematically investigated. The optimized experimental conditions for separation and detection of N-nitrosoamino acids were described.     

Determination of sulphonated azo dyes in food by ion-pair liquid chromatography with photodiode array and electrospray mass spectrometry detection

An ion-pair liquid chromatography method with on-line photodiode-array and electrospray mass spectrometry detection was developed to determine 10 commonly used sulphonated azo dyes (Tartrazine, Amaranth, New Coccine, Sunset Yellow FCF, Allura Red AC, Ponceau R, Ponceau 3R, Orange I, Orange II and Metanil Yellow) in food. A reversed phase C(18) column with gradient elution was utilized to separate these compounds. Triethylamine was added in the mobile phase as an ion-pair reagent for chromatographic separation. Photodiode-array detection was employed for quantitative determination and electrospray mass spectrometry was used for identification. Good linearity (0.05-10 ppm, r(2)=0.999) and detection limit (<0.01 ppm) were determined with 5 mul injection. In addition, precision and accuracy associated with this newly developed method will be presented. A liquid extraction method was also developed to extract these dyes from different foodstuffs. The application of this method was demonstrated by analyzing sulphonated azo dyes in soft drinks, fruit jam, and salted vegetables.     

Analysis of metal complex azo dyes by high-performance liquid chromatography/electrospray ionization mass spectrometry and multistage mass spectrometry

Five metal complex azo compounds were analyzed using negative-ion electrospray ionization mass spectrometry (ESI-MS). Mass spectra of all compounds yield intense peaks corresponding to [M - H](-) ions without any fragmentation, where M denotes the neutral compound with a proton as the counterion. Under collision induced dissociation (CID) conditions, structurally important fragment ions were studied using the ion trap analyzer with a multistage mass spectrometry (MS(n) facility. Synthesized compounds with (15)N atoms in the azo group facilitated the fragmentation pattern recognition. A reversed-phase high-performance liquid chromatography (HPLC) method using 5 mM ammonium acetate in 70% aqueous acetonitrile as mobile phase was developed making possible the separation of all complex compounds tested. The lower detection limits of the ESI-MS method are in the range 10-20 ng of each compound. The HPLC/ESI-MS method makes possible the monitoring of ligand exchange in aqueous solutions of metal complex azo dyes, and also investigation of the stabilities of the complexes in solution. Copyright 2000 John Wiley & Sons, Ltd.     

Liquid chromatographic analysis of nucleosides and their mono-, di- and triphosphates using porous graphitic carbon stationary phase coupled with electrospray mass spectrometry

Analysis of nucleosides and nucleotides is desirable in many biological studies, but the task is analytically challenging due to the high polarity of the analytes. In this study, resolution of mixtures containing nucleosides and their mono-, di- and triphosphates was achieved using a porous graphitic carbon (PGC) stationary phase, Hypercarb, under conditions suitable for liquid chromatography/mass spectrometry (LC/MS). Different organic mobile phases and modifiers were evaluated and the separation of 16 nucleosides and nucleotides was optimized using gradient elution with a water/acetonitrile mobile phase containing ammonium acetate and diethylamine as modifiers. The ammonium acetate concentration proved to be critical for retention and diethylamine was found to improve the peak shapes of di- and triphosphates for mass spectrometric detection. A variety of silica-based columns designed for polar compound separation were also tested using optimized LC conditions and compared with results obtained with the Hypercarb column. Only the Hypercarb column provided separations suitable for accurate quantitation of mixed nucleosides and their phosphates.     

Competitional hydrophobicity driven separations under RP‐LC mechanism: Application to sulfonylurea congeners

Retention of a model set of sulfonylurea compounds has been studied under RP‐LC conditions, considering competitional effects brought by different alcohols (ethanol, 1‐propanol, 2‐propanol, 1‐butanol, 1‐pentanol, and 1‐octanol) used as additives in the organic component of the mobile phase (methanol). The capacity factors determined for the model compounds decreased with the increase of the hydrophobic character of the organic additive in the mobile phase. The amount of the additive within the organic component of the mobile phase was kept constant (1% as volumetric ratio). Retention was studied at different mobile phase compositions (aqueous to organic component ratios). Different functional fitting models were used to correlate retention to the content of the organic component in the mobile phase. Extrapolation of retention expressed as capacity factor to a mobile phase composition free of organic component is well correlated to the hydrophobic characteristics of the organic additives. The adsorption model was used for tuning the experimental find‐outs. The possibility of controlling retention through the competitive effects induced by hydrophobic additives in the mobile phase is highlighted.     

Effect of organic mobile phase composition on signal responses for selected polyalkene additive compounds by liquid chromatography-mass spectrometry

The high performance liquid chromatography (HPLC) separation methodology employed in the study of polyalkene additive compounds by atmospheric pressure ionization mass spectrometry (API-MS) was undertaken. Both atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) were examined. APPI (including dopant-assisted APPI) was found to be an inferior ionization technique to APCI in all cases. APCI ion responses were found to be highly dependent upon the organic solvent type used in the HPLC separations. Namely, employing a water/methanol gradient in place of a water/acetonitrile or a water/acetone gradient yielded improvements in analyte ion intensities between 2.3- and 52-fold for the liquid chromatography-mass spectrometry (LC-MS) experiments. Analyte and mobile phase solvent ionization energies were found to be only partially responsible, whereas mobile phase cluster formation and hydration was also implicated. Mobile phase component modification is demonstrated to be an important consideration when developing new, or modifying existing HPLC separations for use in LC-MS experiments in order to enhance analyte sensitivity for a wide variety of common polyalkene additives.