Addition Of Ion-pair Reagents Research Articles

Quantitation of endogenous nucleoside triphosphates and nucleosides in human cells by liquid chromatography tandem mass spectrometry.

Nucleosides and nucleoside triphosphates are the building blocks of nucleic acids and important bioactive metabolites, existing in all living cells. In the present study, two liquid chromatography tandem mass spectrometry methods were developed to quantify both groups of compounds from the same sample with a shared extraction procedure. After a simple protein precipitation with methanol, the nucleosides were separated with reversed phase chromatography on an Atlantis T3 column while for the separation of the nucleoside triphosphates, an anion exchange column (BioBasic AX) was used. No addition of ion pair reagent was required. A 5500 QTrap was used as analyzer, operating as triple quadrupole. The analytical method for the nucleoside triphosphates has been validated according to the guidelines of the US Food and Drug Administration. The lower limit of quantification values were determined as 10pg on column (0.5ng/mL in the injection solution) for deoxyadenosine triphosphate and deoxyguanosine triphosphate, 20pg (1ng/mL) for deoxycytidine triphosphate and thymidine triphosphate, 100pg (5ng/mL) for cytidine triphosphate and guanosine triphosphate, and 500pg (25ng/mL) for adenosine triphosphate und uridine triphosphate respectively. This methodology has been applied to the quantitation of nucleosides and nucleoside triphosphates in primary human CD4 T lymphocytes and macrophages. As expected, the concentrations for ribonucleosides and ribonucleoside triphophates were considerably higher than those obtained for the deoxy derivatives. Upon T cell receptor activation, the levels of all analytes, with the notable exceptions of deoxyadenosine triphosphate and deoxyguanosine triphosphate, were found to be elevated in CD4 T cells.

Rapid quantitative analysis of carnitine and acylcarnitines by ultra-high performance–hydrophilic interaction liquid chromatography–tandem mass spectrometry

l-Carnitine and its acyl esters (acylcarnitines) play an important role in the metabolism of fatty acids. However, most of the present methods for the quantitative analysis of acylcarnitines have restrictions both in sample preparation and in chromatographic separation. Herein we present a validated method for determination of carnitine and eleven acylcarnitines in human serum and rat tissue biopsies by using ultra-high performance–hydrophilic interaction liquid chromatography–tandem mass spectrometry (UHP–HILIC–MS/MS). The procedure uses minimal sample preparation including only addition of organic solvent, labeled internal standard, incubation and centrifugation. The separation is performed without derivatization or addition of ion-pairing reagent within 7min on a hydrophilic interaction liquid chromatographic column with mass spectrometric detection. The method is linear in response over the concentration range from 20 to 600ng/ml for carnitine and acetylcarnitine and 5–200ng/ml for the other acylcarnitines, with correlation coefficients higher than 0.994. Recoveries were higher than 88% for most of the compounds. Limits of detection were 5ng/ml for carnitine and acetylcarnitine and approximately 0.5ng/ml for other acylcarnitines. The method was applied to the analysis of serum and tissue samples.

Teresa Cecchi: Ion-pair chromatography and related techniques

Teresa Cecchi: Ion-pair chromatography and related techniques

Determination of melamine in milk powder using zwitterionic HILIC stationary phase with UV detection

A hydrophilic interaction liquid chromatography-UV method was developed for the determination of melamine in milk powder. Sulfobetaine type zwitterionic hydrophilic interaction liquid chromatography stationary phase was used to achieve straightforward separation of melamine in milk powder without any sample derivatization or addition of ion-pair reagent. The sample preparation was simple and fast with the steps of acetonitrile/perchloric acid extraction-centrifugation-filtration. No SPE or other pre-concentration procedure was required. By using large volume sample injection and choosing low UV wavelength (210 nm), the LOD and LOQ were 0.005 and 0.015 microg/mL for melamine standards, and 0.02 and 0.06 microg/mL for the spiked milk extracts. The LOD and LOQ of the latter correspond to 0.95 and 2.2 microg/g melamine in the milk powder. The correlation coefficients for melamine standard, pre-spiked milk extracts and post-spiked milk extracts in the range of 0-0.5 microg/mL were 0.9978, 0.9976 and 0.9995, respectively. This newly developed method is sensitive and cost effective, therefore, suitable for screening of melamine in tainted milk products.

Identification and Quantification of Glycoproteins Using Ion-Pairing Normal-phase Liquid Chromatography and Mass Spectrometry

Glycoprotein structure determination and quantification by MS requires efficient isolation of glycopeptides from a proteolytic digest of complex protein mixtures. Here we describe that the use of acids as ion-pairing reagents in normal-phase chromatography (IP-NPLC) considerably increases the hydrophobicity differences between non-glycopeptides and glycopeptides, thereby resulting in the reproducible isolation of N-linked high mannose type and sialylated glycopeptides from the tryptic digest of a ribonuclease B and fetuin mixture. The elution order of non-glycopeptides relative to glycopeptides in IP-NPLC is predictable by their hydrophobicity values calculated using the Wimley-White water/octanol hydrophobicity scale. O-linked glycopeptides can be efficiently isolated from fetuin tryptic digests using IP-NPLC when N-glycans are first removed with PNGase. IP-NPLC recovers close to 100% of bacterial N-linked glycopeptides modified with non-sialylated heptasaccharides from tryptic digests of periplasmic protein extracts from Campylobacter jejuni 11168 and its pglD mutant. Label-free nano-flow reversed-phase LC-MS is used for quantification of differentially expressed glycopeptides from the C. jejuni wild-type and pglD mutant followed by identification of these glycoproteins using multiple stage tandem MS. This method further confirms the acetyltransferase activity of PglD and demonstrates for the first time that heptasaccharides containing monoacetylated bacillosamine are transferred to proteins in both the wild-type and mutant strains. We believe that IP-NPLC will be a useful tool for quantitative glycoproteomics.

Optimization of Chromatographic Systems for Determination of Lipophilicity for Selected Isoquinoline Alkaloids

Nine isoquinoline alkaloids were investigated on C18 stationary phase in different eluent systems containing acetonitrile, methanol, tetrahydrofuran, and dioxane as organic modifier with addition of buffers at different pH, ion pairing reagents, or silanol blockers. The dependencies between the concentration of organic modifier and retention parameters were plotted. Log kw values were determined by the extrapolation method. The values, obtained in different eluent systems, were correlated with a predicted lipophilicity parameter (log P). The correlations between chromatographic values and predicted lipophilicity values are different, depending on the eluent system applied and the estimation method concerning partition coefficients: octanol/water. The best correlations were obtained for eluents containing the addition of ion pair reagents, especially for systems containing the sodium dodecyl sulfate.

High performance liquid chromatographic measurement of iothalamate in human serum and urine for evaluation of glomerular filtration rate

A simple and sensitive HPLC-UV assay was developed for the measurement of iothalamate (IOT) in human serum and urine. Chromatographic separation was achieved using an embedded-carbamate-group bonded RP18 column and mobile phase consisting of 50 mM monobasic sodium phosphate and methanol (90:10, v/v) without the addition of ion-pair reagents. The assay demonstrated a high analytical reliability within the IOT concentration range of 1–150 μg/ml in serum and 25–1500 μg/ml in urine. The relative standard deviations (RSDs) for intra- and inter-day analysis were less than 5.1% in all cases. This method has been used for the evaluation of glomerular filtration rate (GFR) in subjects participating in a phase I clinical trial of a novel antimalarial medicine. The average baseline GFR was 100.41 ± 19.99 ml/min/1.73 m 2 in 119 healthy volunteers. The assay may also allow the simultaneous measurements of p-aminohippuric acid (PAH), N-acetyl PAH (aPAH), and IOT with some modification. PAH, IOT, aPAH, and β-hydroxyethyl-theophylline internal standard peaks appeared approximately at 2.5, 3.7, 5.9, and 11.8 min, respectively, in an isocratic run.

On-line sample preconcentration in micellar electrokinetic chromatography using ion-pair reagents.

To improve the detection sensitivity of some aromatic carboxylic acids and naphthalenesulfonic acids, the use of ion-pair reagents was examined in sweeping micellar electrokinetic chromatography (MEKC) with an anionic sodium dodecyl sulfate (SDS) micelle. Tetraalkylammonium (TAA) salts were used as ion-pair reagents to improve the sweeping efficiency. The effects of the alkyl chain length of the TAA groups and the TAA salt concentration on sweeping were examined. Under optimized conditions, about 400-fold enhancement in detection sensitivity was obtained in terms of peak heights by addition of ion-pair reagents in sweeping MEKC. This value was about 10 times greater than that obtainable by the SDS micelle used alone.

APPLICATION OF SMALL CAPACITY CATION EXCHANGERS IN ION-EXCLUSION CHROMATOGRAPHY

Different attempts to increase retention of strong acids in ion exclusion chromatography are presented. A decrease of the dissociated forms of the sorbent functional groups which increases the retention of strong acids can be achieved either by decreasing the effective charge of the support, or by using a week exchanger as silica, instead of conventionally sulfonated resins. Another suggested way is the addition of ion pairing reagents to the mobile phase. All these attempts, which improve the separations, fit with a proposed theoretical model.

High-performance liquid chromatographic analysis of chlorhexidine and p-chloroaniline using a specialty column and a photodiode-array detector

A high-performance liquid chromatographic method has been developed for the separation of chlorhexidine and its known degradation product, p-chloroaniline. These amine-containing compounds can be separated without the addition of ion-pairing reagents and/or amine modifiers if the proper specialty column is selected. A photodiode-array detector was used to acquire spectral data and demonstrate the importance of the mobile phase pH when optimizing the response of p-chloroaniline.

Separation, identification and determination of alkyl- and alkylphenol ether carboxylates by HPLC

A method is described which allows the separation, identification and determination of alkyl- and alkyl-phenol ether carboxylates even in mixtures with other anionic, non-ionic or amphoteric surfactants in one step. The separation is carried out by HPLC using a reversed phase RP 18 column and a mixture of methanol, water and acetonitrile with addition of ion pairing reagent as mobile phase working under isocratic conditions. For the detection and quantitative determination a differential refractometer can generally be used. The detection limit is about 10 μg/ml. If only alkylphenol ether carboxylates are to be determined an UV-spectrophotometer can also be used by which the detection limit is lowered with a factor 100 to about 0.1 μg/ml.

Determination of dihydroxyphenylalanine and dihydroxyphenylacetic acid in biological samples by coupled-column liquid chromatography with dual coulometric—amperometric detection

Adrenaline, noradrenaline, dopamine and dihydroxyphenylalanine in brain tissue and dihydroxyphenylacetic acid in plasma have been determined by direct injection of brain homogenates or plasma into a liquid chromatographic system comprising three columns, one packed with a boronic acid gel and two with reversed-phase material. The catecholamines and dihydroxyphenylalanine were selectively adsorbed on the boronic acid gel and separated by ion-pair chromatography on the reversed-phase columns. DOPAC was determined in a modified system without the addition of ion-pairing reagents. The catechols were detected by coulometry or amperometry with two working electrodes, operated in the oxidative—reductive mode. The reductive signal was used for quantification. The limits of determination were 0.05 nmol g −1 with 40 mg brain tissue and 2 nmol 1 −1 with 0.5 ml plasma for DOPA and DOPAC respectively. The limit of quantification of DA and DOPAC in urine was about 0.1 μmol l −1.

Quantification of Suramin by Reverse-Phase Ion-Pairing High-Performance Liquid Chromatography

Abstract A specific and sensitive method has been developed for the separation and quantification of suramin and trypan blue (internal standard) in human plasma. Plasma samples were extracted by centrifugation after the addition of ion-pairing reagent (tetra-butylammonium phosphate, TRAP) and methanol. Extracts were injected directly onto a reverse-phase ion-pairing HPLC system with 5 mM TBAP in the mobile phase. There was nearly 100% extraction efficiency after 3 cumulative extracts of each sample. The limit of quantitation was 0.5 μg/ml at a detection wavelength of 313 nm. Analysis of 3 post-therapy samples from a patient with AIDS was used to determine a plasma half-life for suramin of at least 3 weeks.