Determination of 20 underivatized proteinic amino acids by ion-pairing chromatography and pneumatically assisted electrospray mass spectrometry.

A qualitative determination of 20 underivatized proteinic amino acids by LC-MS is reported. The need for chromatographic separation before mass spectrometry determination is demonstrated based on the study of several amino acid pairs which have some similar characteristics. Two suitable LC-MS systems are proposed for amino acid analysis. A preliminary optimization of these systems has been investigated using evaporative light scattering detection as these two detection modes have the same chromatographic requirements. The amino acid separation was achieved on a Purospher RP-18e or a Supelcosil ABZ+Plus column with tridecafluoroheptanoic acid or pentadecafluorooctanoic acid as volatile ion-pairing reagent in an acetonitrile-water mobile phase. In order to elute the most retained amino acids, an elution gradient based on simultaneously increasing the concentration of acetonitrile and decreasing the concentration of the ion-pairing reagent was used. The detection limits of the present work (without specialized optimization) varied from 0.5 to 1 mg 1(-1).     

Speciation of arsenical species by anion-exchange and ion-pair reversed-phase liquid chromatography

Summary Speciation and quantitative analysis of arsenical compounds are performed by using high-performance liquid chromatography (HPLC) with direct UV detection. Ion chromatography has been used to separate mixtures of arsenical compounds (arsenite, MMA, DMA, arsenate) on an anion-exchange column using phosphate buffer (1 mmol/l, pH=5.3) as eluent. Ion -pair reversed-phase chromatography has been investigated to resolve mixtures of arsenite, arsenate, MMA, DMA, arsenobetaine and arsenocholine on an octadecyl-bonded silica column using water as mobile phase (pH=7.3) and tetrabutylammonium cation as ion-pairing reagent. The influence of several parameters (pH, the ion-pairing reagent concentration or the amount of methanol in the mobile phase) has been studied to determine the best chromatographic conditions.     

Regeneration of tetrabutylammonium ion-pairing reagent distribution in a gradient elution of reversed phase ion-pair chromatography

The regeneration of ion-pairing reagent distribution on liquid chromatography columns after gradient elution has been well recognized as the cause for long column equilibration time, a major drawback associated with gradient elution reverse phase ion-pair chromatography. To date, the majority of studies have focused on optimizing the separation conditions to shorten the equilibration time. There is limited understanding of the ion-pairing reagent distribution process between the mobile phase and stationary phase in the course of gradient elution, and subsequent column re-equilibration. The focus of this work is to gain a better understanding of this process. An ion-pair chromatographic system, equipped with a YMC ODS C(18) column and a mobile phase containing tetrabutylammonium (TBA) hydroxide as the ion-pairing reagent, was used in the study. The TBA distribution profile was established by measuring its concentration in the eluent fractions collected during the gradient cycle using different column equilibration times with an ion chromatographic method. Furthermore, the analyte retention time was evaluated as the function of the column equilibration time and TBA concentration in the mobile phase. The column equilibration and its impact on the method robustness will also be discussed.     

A systematic study of the desorption of different ion-pairing reagents on reversed phase thin-layers

Summary The absolute concentrations and the concentration distribution of different cationic ion-pairing reagents, such as tetramethylammonium bromide, tetrabutylammonium bromide, cetyltrimethylammonium bromide and trioctylmethylammonium chloride were studied after chromatographic development on physically and chemically bonded reversed-phase layers. The combination of the chromatographic conditions involved three variations. Untreated layers were chromatographed with mobile phases containing the ion-pairing reagent. Several layers were treated with the ion-pairing reagent prior to the chromatographic run, and methanol-water mixtures were used as mobile phase. In a third set of experiments both the layer and the mobile phase contained the ion-pairing reagent. The chromatographic behaviour of acid, neutral and basic model compounds were also studied. For the determination of different reagents remaining on the layer after the chromatographic run, spectrophotometric, potentiometric methods and capillary electrophoresis were used. The performance of the analytical methods was evaluated. Results obtained for the absolute concentrations and distribution of the reagents and the retention data were compared to those obtained on silica gel layers. A discussion of the retention mechanism is given. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

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

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

Strategies for the Analysis of Pharmaceutical Cocrystals Using HPLC with Charged Aerosol Detection

Several active pharmaceutical ingredients are currently being developed as pharmaceutical cocrystals as these systems often have superior properties compared to traditional pharmaceutical forms. Pharmaceutical cocrystal formers typically used are polar, small molecule acids or bases which often lack a UV chromophore. Their polar nature results in almost no reversed phase retention and their detection typically cannot be done with UV. Here we discuss approaches for the analysis of pharmaceutical cocrystals using HPLC columns designed for polar retention, ion pairing chromatography (IPC), and hydrophilic interaction chromatography (HILIC) using model cocrystal formers. Corona charged aerosol detection (CAD) was used to monitor the cocrystal formers. l-alanine was used as a model basic cocrystal former, and succinic acid and glutaric acid were used as model acidic cocrystal formers. The acidic cocrystal formers were adequately retained on a C18 column. Heptafluorobutyric acid was used as the ion-pairing reagent for l-alanine as it was unretained without the ion-pairing reagent. HILIC, a newer approach for polar compound retention, was also investigated. Using the HILIC mode, all three model cocrystal formers were retained adequately. Of all the approaches studied for the analysis of the cocrystal formers, HILIC appears to be the best choice as the same column can be used for both acidic and basic cocrystal formers. With IPC, the ion-pairing reagent permanently alters the column chemistry and dedicated columns are required for each ion-pairing reagent used. CAD detection provided a linear response in the 80–100% test concentration range for the analytes studied here.     

Optimization of the determination of inorganic and organic selenium species using high-performance liquid chromatography-electrothermal atomic absorption spectrometry

The liquid chromatographic simultaneous separation of selenite, selenate, selenocystine and selenomethionine was studied with a selenium-specific detector; electrothermal atomic absorption spectrometry. Three chromatographic modes were compared; two ion-pairing ones using either a cationic or an anionic reagent and ion-exchange. Separation parameters such as composition, pH and concentration of the mobile phase were investigated. The influence of chromatographic conditions on the sensitivity of the detector is studied. The ion-exchange method was determined to give the best results, with detection limits ranging from 8 to 17 μg l⁻¹ within a 30 min separation time. An application of this method to the analysis of seleno compounds present in a selenium-rich yeast after enzymic hydrolysis extraction is presented.     

Reversed phase ion-pair high performance liquid chromatographic gradient separation of related impurities in 2,4-disulfonic acid benzaldehyde di-sodium salt

A reversed phase ion-pair gradient liquid chromatographic method has been developed and validated for purity determination of the hydrophilic compound 2,4-disulfonic acid benzaldehyde di-sodium salt (2,4-DSAD) containing both hydrophilic and more lipophilic related impurities. Mixtures of acetonitrile-phosphate buffer containing tetrahexylammonium hydrogen sulfate as the ion-pairing reagent were used as the mobile phase. A linear gradient, which generated simultaneous change in the concentration of organic modifier, buffer concentration and the concentration of ion-pairing reagent, was applied. The method allows detection of impurities at low levels (0.01% w/w). Excellent repeatability for both retention time (RSD< or =0.3%, n = 6) and detector response (RSD = 0.03%, n = 6 for the main peak and RSD = 6%, n = 6, for an impurity at 0.01 area% level) was obtained. The method was shown to be robust for routine analysis and has been successfully transferred to the quality control laboratory.     

Selective, stability-indicating assay of the major ipecacuanha alkaloids, emetine and cephaeline, in pharmaceutical preparations by high-performance liquid chromatography using spectrofluorimetric detection

A selective high-performance liquid chromatographic procedure has been developed for the determination of the major Ipecacuanha alkaloids, emetine and cephaeline, in a number of linctus and pastille preparations. The reversed-phase chromatographic procedure uses an octadecyl-bonded column with a mobile phase of aqueous methanol containing an ion-pairing reagent. A spectrofluorimetric detector is used for increased sensitivity and selectivity. Sample preparation is simple, involving either straight dilution for linctus formulations or simple dissolutions for pastilles. The procedure has been shown to be stability-indicating. Validation studies, to show that the method is precise, accurate and rectilinear, have been carried out on four linctus formulations and two pastille formulations. The method has been used to determine both emetine and cephaeline at levels as low as 5 micrograms/g in formulations.     

Positive mode electrospray ionization mass spectrometry of bisphosphonates using dicationic and tricationic ion-pairing agents

A general method for detecting bisphosphonate drugs by ESI-MS and LC-ESI-MS as positive ions has been developed. Bisphosphonates can have multiple negative charges in solution. Tricationic ion-pairing reagents were paired with bisphosphonates to form a positively charged complex. It was clear that this facile pairing method worked. However, an appreciable presence of −1 bisphosphonate species were observed in positive mode ESI-MS (i.e. as the +2 complex with tricationic reagents). This led to an extended investigation on the use of dicationic pairing agents. The use of dicationic reagents improved the detection sensitivity for all of the bisphosphonates. Tandem mass spectrometry also improved the limits of detection for most of the bisphosphonates using both the tricationic and dicationic pairing reagents. A tricationic reagent also was used as an ion-pairing reagent in chromatography experiments. Thus the addition of a single reagent produced benefits in that it increased chromatographic retention and enhanced the ESI-MS detection of bisphosphonates.     

Synergistic effects of ion-pairing in the enantiomeric separation of basic compounds with cyclodextrin derivatives in nonaqueous capillary electrophoresis

The enantiomeric separation of various kinds of basic pharmaceuticals has been investigated in nonaqueous capillary electrophoresis (NACE) systems using an ion-pairing reagent in combination with cyclodextrins (CDs). The simultaneous addition to the methanolic background electrolyte (BGE) of (+)-S-camphorsulfonate or alkanesulfonates and an anionic beta-cyclodextrin derivative, heptakis(2,3-dimethyl-6-sulfato)-beta-cyclodextrin (HDMS-beta-CD), led to partial or complete enantioresolution in most cases. In the absence of ion-pairing reagent, the enantiomeric resolution obtained with this CD derivative was most often completely lost or strongly reduced, indicating the important role of ion-pairing in the chiral recognition mechanism in these NACE systems. The influence of the nature and concentration of the counterion and the anionic CD derivative on the enantioseparation of basic compounds was studied. Synergistic effects between these two kinds of charged additives were clearly observed.     

High-performance ion-pair chromatography method for simultaneous analysis of alliin,deoxyalliin, allicin and dipeptide precursors in garlic products using multiple mass spectrometry and UV detection

The quality of garlic and garlic products is usually related to their alliin content and allicin release potential. Until now no analytical method was able to quantify simultaneously allicin, its direct precursor alliin (S-allyl-L-cysteine sulfoxide), SAC (S-allyl-L-cysteine) as well as various dipeptides that apparently serve as storage compounds in garlic. It is well known that all these intermediates are involved in the allicin biosynthetic pathway. A simple and rapid HPLC method suitable for routine analysis was developed using eluents containing an ion-pairing reagent. Particularly, heptanesulfonate as ion-pairing reagent guarantees a sufficient separation between alliin and the more retained dipeptides at very low pH. Allicin was eluted after 18 min on a 150 x 3 mm column. Synthetic reference compounds were characterized by the same chromatographic method using a diode-array UV detector and an ion trap mass spectrometer (electrospray ionization) in the multiple MS mode. In routine analysis of garlic bulbs, powders and other products, the diode-array detector is sufficient for a relevant quantification. Our method has been used in studies to improve the quality of garlic and its derived products.     

A systematic approach toward comparing electrospray ionization efficiencies of derivatized and non‐derivatized amino acids and biogenic amines

Ionization efficiency (IE) in mass spectrometry (MS) has been studied for many different compounds, and different IE scales have been constructed in order to quantitatively characterize IE. In the case of MS, derivatization has been used to increase the sensitivity of the method and to lower the limits of detection. However, the influence of derivatization on IE across different compounds and different derivatization reagents has not been thoroughly researched, so that practitioners do not have information on the IE‐enhancing abilities of different derivatization reagents. Moreover, measuring IE via direct infusion of compounds cannot be considered fully adequate. Since derivatized compounds are in complex mixtures, a chromatographic method is needed to separate these compounds to minimize potential matrix effects. In this work, an IE measurement system with a chromatographic column was developed for mainly amino acids and some biogenic amines. IE measurements with liquid chromatography electrospray ionization mass spectrometry (LC/ESI/MS) were carried out, and IE scales were constructed with a calibration curve for compounds with and without derivatization reagent diethyl ethoxymethylenemalonate. Additionally, eluent composition effects on ionization were investigated. Results showed that derivatization increases IE for most of the compounds (by average 0.9 and up to 2‐2.5 logIE units) and derivatized compounds have more similar logIE values than without derivatization. Mobile phase composition effects on ionization efficiencies were negligible. It was also noted that the use of chromatographic separation instead of flow injection mode slightly increases IE. In this work, for the first time, IE enhancement of derivatization reagents was quantified under real LC/ESI/MS conditions and obtained logIE values of derivatized compounds were linked with the existing scale.     

Divalent pairing ion for ion-interaction chromatography of sulphonates and car☐ylates

An ion-interaction method for the simultaneous separation and UV detection of compounds with weak or strong ionic groups, using a divalent cationic ion pair reagent, namely hexamethonium bromide, has been studied and developed. The analytes considered were tartaric, fumaric, pyruvic, maleic, phthalic, benzoic, sorbic, 4-hydroxybenzoic, benzene and toluene-4-sulphonic acids, and they were chosen for their practical importance. The chromatographic optimization for their separation has been achieved by varying eluent composition (methanol, NaCl and hexamethonium concentrations) and studying the performance of different stationary phases (octyl- and octadecyl-silica based columns). The method developed has been successfully applied to benzoate and sorbate determination in orange juices.     

Analysis of phosphorus herbicides by ion-pairing reversed-phase liquid chromatography coupled to inductively coupled plasma mass spectrometry with octapole reaction cell

A reversed phase ion-pairing high performance liquid chromatographic (RPIP-HPLC) method is developed for the separation of two phosphorus herbicides, Glufosinate and Glyphosate as well as Aminomethylphosphonic acid (AMPA), the major metabolite of Glyphosate. Tetrabutylammonium hydroxide is used as the ion-pairing reagent in conjunction with an ammonium acetate/acetic acid buffering system at pH 4.7. An inductively coupled plasma mass spectrometer (ICP-MS) is coupled to the chromatographic system to detect the herbicides at m/z = 31P. Historically, phosphorus has been recognized as one of the elements difficult to analyze in argon plasma. This is due to its relatively high ionization potential (10.5 eV) as well as the inherent presence of the polyatomic interferences 14N16O1H+ and 15N16O+ overlapping its only isotope at m/z = 31. An octapole reaction cell is utilized to minimize the isobaric polyatomic interferences and to obtain the highest signal-to-background ratio. Detection limits were found to be in the low ppt range (25-32 ng/l). The developed method is successfully applied to the analysis of water samples collected from the Ohio River and spiked with a standard compounds at a level of 20 microg/l.     

2-Bromoacetyl-6-methoxynaphthalene: A useful fluorescent labelling reagent for HPLC analysis of carboxylic acids

Summary The use of 2-bromoacetyl-6-methoxynaphthalene as a fluorogenic labelling reagent in pre-column derivatization for the HPLC separation of biologically active carboxylic acids (fatty acids and bile acids) has been investigated. The compound reacts (30 min. at 70°C) with carboxylic acids to give fluorescent esters that can be separated by reversedphase HPLC and detected at ex. 300 nm, em. 460 nm. The experimental conditions for the derivatization and chromatographic separation are discussed. Applications to the determination of valproic acid and chenodeoxycholic acid in pharmaceutical formulations are described.     

Preparation and application of a new ligand exchange chiral stationary phase for the liquid chromatographic resolution of alpha-amino acid enantiomers

A new liquid chromatographic ligand exchange CSP has been prepared by covalently bonding (S)-N,N-carboxymethyl undecyl leucinol monosodium salt onto silica gel and employed in resolving various alpha-amino acids. The new CSP was quite good in resolving various a-amino acids and the resolution results were dependent on the type and content of organic modifier in the mobile phase. From these results, a chiral recognition model using a lipophilic interaction between the tethering alkyl group of the CSP and the substituent at the chiral center of alpha-amino acids was proposed. The liquid chromatographic resolution of alpha-amino acids on the new CSP was also found to be dependent on the Cu(II) concentration in the mobile phase and the column temperature.     

Reversed-Phase Ion-Pairing Liquid Chromatographic Separation and Fluorimetric Detection of Polyamines

Abstract

A rapid and specific reversed-phase ion-pairing high performance liquid chromatographic procedure for putrescine, spermidine and spermine is reported. The ion-pairing reagent, heptanesulfonate, was employed and o-phthalaldehyde and 2-mercaptoethanol were used for on-line post-column derivatization and subsequent fluorescence detection. Experiments were carried out to determine the effects of several variables such as pH, concentration of the aqueous buffer, counter-ion concentration, and the percentage of organic modifier in the moving phase. The minimum detection limits for the polyamines ranged from 120 pmoles for spermine to 12 pmoles for putrescine. The method includes a gradient program which provides complete separation from amino acids and specificity for the three polyamines. The procedure was applied successfully to urine and serum samples.     

Ion-pairing high-performance liquid chromatographic method for the determination of 5-aminosalicylic acid and related impurities in bulk chemical

An ion-pairing high-performance liquid chromatographic method has been developed for the determination of 5-aminosalicylic acid (5-ASA) bulk chemical in the presence of thirteen potential synthetic process impurities. In addition, the method is suitable for the determination of the in process intermediate, 5-nitrosalicylic acid. A selective method was achieved on a Hypersil-BDS reversed-phase column using 1-heptanesulfonic acid sodium salt as the ion-pairing reagent in a 0.08 M sodium phosphate buffer (pH 2) containing 0.005 M 1-heptanesulfonic acid sodium salt and 0.07 M sodium chloride-methanol-tetrahydrofuran (85:11:4, v/v/v) isocratic mobile phase. The method was validated using a multi-day, intra-laboratory protocol. The validation addressed linearity, accuracy, precision, sensitivity, and ruggedness of the method. The validated method characterizes the purity of 5-ASA bulk chemical.