Effect Of Mobile Phase Additives Research Articles

Ion-pair chromatographic attribute in the retention of ammonium ion on a crown ether-based and a polysaccharide-based chiral stationary phases. Some mobile phase additives compatible with liquid chromatography-electrospray ionization-mass spectrometry

The design of the mobile phase additive useful in HPLC analysis on two types of chiral stationary phases, one based on chiral crown ether, and another based on polysaccharide derivative, was developed. CROWNPAK® CR-I, the chiral stationary phase bearing (R)- or (S)-(3,30-diphenyl-1,10-binaphtyl)-20-crown-6 (1) moiety is a useful CSP to separate primary amine enantiomers. While perchloric acid had been recommended as the mobile phase additive to attain a better separation, we have reexamined the effect of mobile phase additives and revealed that the retention of an ammonium ion is enhanced by a chaotropic or a hydrophobic anion in a water-based mobile phase like well-known ion pair chromatography in an RPLC. However, the retention was enhanced in reverse by a rather cosmotropic or a hydrophilic anion in an acetonitrile (ACN)-based mobile phase. These results looking contradictory to each other may be rationalized both in the basis of ion pair chromatography. A similar but smaller effect of pairing anion species was also observed on a polysaccharide-based CSP. Based on these findings, some types of mobile phase have been proposed and some of them were demonstrated to be compatible with liquid chromatography-electrospray ionization-mass spectrometry (ESI LC/MS).

Chiral LC method development: Stereo-selective separation, characterization, and determination of cabotegravir and related RS, RR, and SS isomeric impurities on coated cellulose-based chiral stationary phase by HILIC-LC and LC-MS

The first, sensitive, and rapid chiral method was developed for enatioseperations and determination of cabotegravir and its enantiomeric impurities by using HPLC and LC-MS. Cabotegravir is an antiretroviral medication used for the treatment of HIV/AIDS approved by the food and drugs administration (FDA) in the year 2021. The cabotegravir chiral separation was achieved on the coated cellulose-tris (4-chloro-3-methyl phenyl carbamate) (CHIRALCEL OX-3R) column in HILIC mode and the total run time is less than 15 min. The effects of mobile phase composition, elution mode, and percentage of organic modifier as well as the effect of mobile phase-additives and column temperature were investigated on selectivity, resolution, and peak symmetry. The mobile phase consisted of acetonitrile and water with 0.1% (v/v) addition of formic acid additive with the flow rate of 1 mLmin−1. UV detection was carried out at 220 nm. The calibration curves of cabotegravir and its enantiomers were linear over the concentration range of 0.04–1.125 µgmL−1. The limits of detection and quantification for cabotegravir and its enantiomer (RS isomer) were ≤ 0.02 and ≤ 0.06, and the RR and SS-isomers limits were ≤ 0.02 and 0.03 µgmL−1 respectively. It was demonstrated that the proposed method is selective, precise, and robust. Finally, the validated method was applied for the determination and identification of cabotegravir and its chiral enantiomers in the bulk drugs by using HPLC and LC-MS techniques.

Unravelling the effects of mobile phase additives in supercritical fluid chromatography—Part II: Adsorption on the stationary phase

The mobile phases employed in current practice of supercritical fluid chromatography (SFC) are usually composed of a mixture of pressurized carbon dioxide and a co-solvent. The co-solvent is most often an alcohol and may contain a third component in small proportions, called an additive (acid, base or salt). In the first part of this series, the effects of mobile phase additives on the polarity and apparent pH of the mobile phase were explored. In the present paper, we examine the effects pertaining to adsorption of additives on the stationary phase. Ammonium acetate was selected as a representative case, because it is often employed in current practice. To favour its solubility and further improve chromatographic quality, a small portion of water is also advocated. First, the equilibration time is observed to be largely increased in the presence of an additive, especially when mobile phase compositions containing only low proportions of methanol co-solvent are employed. Secondly, the effects of ammonium acetate are more thoroughly assessed with a modified version of the solvation parameter model (five Abraham descriptors and two descriptors to take account of positive and negative charges on ionizable species). On a hybrid silica stationary phase (ACQUITY UPC2 BEH), the effects of increasing concentration of ammonium acetate (0–25 mM in the methanol co-solvent) are investigated. The retention of acidic species is the most strongly affected, with a continuous retention increase when additive concentration increases. The retention of basic and neutral species is also moderately affected. Then thirty-two stationary phases based on sub-2 μm totally porous particles or sub-3 μm superficially porous particles are characterized with and without 20 mM ammonium acetate and 2% water in the methanol co-solvent. The effects of adsorbed additive on the interaction capabilities are discussed. Finally, the interest of introducing the additive in the dilution solvent, a method that was sometimes recommended to simplify the workflow, is also discussed with a model basic compound.

Unravelling the effects of mobile phase additives in supercritical fluid chromatography. Part I: Polarity and acidity of the mobile phase

The mobile phases employed in current supercritical fluid chromatography (SFC) are usually composed of a mixture of pressurized carbon dioxide and a co-solvent. The co-solvent is most often an alcohol and may contain a third component in small proportions, called an additive (acid, base or salt). The polarity of such mobile phase compositions is here re-evaluated with a solvatochromic dye (Nile Red), particularly to assess the contribution of additives. It appears that additives, when employed in usual concentration range (0.1% or 20mM) do not modify the polarity in the immediate environment of the probe.In addition, the combination of carbon dioxide and an alcohol is known to form alkoxylcarbonic acid, supposedly conferring some acidic character to SFC mobile phases. Direct measurements of the apparent pH are impossible, but colour indicators of pH can be used to define the range of apparent pH provided by carbon dioxide-alcohol mixtures, with or without additives. Five colour indicators (Thymol Blue, Bromocresol Green, Methyl Red, Bromocresol Purple, and Bromothymol Blue) were selected to provide a wide range of aqueous pKa values (from 1.7 to 8.9). UV–vis absorption spectra measured in liquid phases of controlled pH were compared to those measured with a diode-array detector employed in SFC, with the help of chemometric methods. Based on these observations, it is concluded that the apparent pH range in carbon dioxide-methanol mobile phases is close to 5. Increasing the proportion of methanol (in the course of a gradient elution for instance) causes decreasing apparent pH. Strong acids can further decrease the apparent pH below 1.7; strong bases have little influence on the apparent pH, probably because, in this range of concentrations, they are titrated by alkoxylcarbonic acid or form ion pairs with alkoxycarbonate. However, bases and salts could stabilize the acidity in the course of gradient runs.

Effect of mobile phase additives on solute retention at low aqueous pH in hydrophilic interaction liquid chromatography

Trifluoracetic acid (TFA) added to the aqueous acetonitrile mobile phase induces some unexpected changes in the ionic component of retention in hydrophilic interaction separations when using Type B silica and amide-bonded silica columns. TFA use results in anion exchange properties which contrast with the cation exchange typically found with ammonium salt buffers. The significant cation exchange properties of silica hydride columns are also moderated by TFA. Similar behaviour was shown in a metal- free amide column operated on a system washed with a metal complexing agent, suggesting that adsorbed metal cations were not responsible for this anion exchange behaviour. Both suppression of silanol ionisation at low pH and ion pairing of bases with TFA could contribute to this effect. It is also possible that the column surface acquires some positive charges at the low pH of TFA. A surprising reversal of the properties of the columns back to predominately cation exchange behaviour was shown using methanesulfonic acid (MSA), which appears to be a stronger acid than TFA in high concentrations of acetonitrile. MSA maintains sufficient ionic strength in the mobile phase even at low concentrations, giving good peak shape, which could be useful for mass spectrometry detection. Besides giving different selectivity to TFA, MSA also gives different selectivity to that of ammonium salt buffers, suggesting it may be useful in manipulating the selectivity of a separation. Similar changes to the selectivity with TFA could be achieved by adding neutral methylsulfonate salts to the TFA mobile phase. While it is possible that methylsulfonate ions are retained on the stationary phase surface, experiments using ion pair reagents of opposite charge yielded the same results as MSA salts. It therefore seems more likely that the higher ionic strength of these solutions negates the influence of charges that may be formed in TFA solutions. Ion pairing effects with MSA are expected to be limited.

Deiodination of iodinated aromatic compounds with electrospray ionization mass spectrometry

Dehalogenation of iodinated X-ray contrast media (ICM) has been reported using electrochemical and bioelectrochemical systems. Correspondingly, dehalogenation of aromatic halogens has also been reported in mass spectrometry (MS) using different ionization techniques like chemical ionization (CI), thermospray, fast-atom bombardment (FAB) and FAB-liquid secondary ionization mass spectrometry (LSIMS). The aim of the present work was to study deiodination of iodinated aromatic compounds in MS with electrospray ionization (ESI). The iodinated aromatic compounds were characterized by liquid chromatography/tandem mass spectrometry (LC/MS/MS) using a quadrupole time-of-flight (QTof)-micro MS instrument and ESI in both positive and negative ion mode. The effect of mobile phase additives like formic acid, acetic acid, trifluoroacetic acid, ammonium formate and ammonium acetate on the negative and positive ESI mass spectra of the iodinated aromatic compounds was studied. Formic acid and ammonium formate induced deiodination of the iodinated aromatic compounds with ESI-MS. Neither acetic acid, trifluoroacetic acid nor ammonium acetate induced the deiodination reaction. The effect was most pronounced with negative ESI where the HI product of the deiodination reaction easily adhered to the aromatic compounds giving rise to HI adducts in the mass spectra. The deiodination reaction was shown to take place in the ESI capillary, since the extent of the reaction was largely dependent on the capillary voltage. The calculated heat of reaction for deiodination of the iodinated aromatic compounds was significantly exothermic for formic acid. This was not the case for acetic acid and trifluoroacetic acid. Care should be taken when using formic acid as a mobile phase additive in LC/MS analyses of iodinated aromatic compounds, since the interpretation of the mass spectra might be influenced by potential dehalogenation reactions taking place in the ESI capillary.

Effect of mobile phase additives on qualitative and quantitative analysis of ginsenosides by liquid chromatography hybrid quadrupole-time of flight mass spectrometry

This study was to systematically investigate the effect of mobile phase additives, including ammonia water, formic acid, acetic acid, ammonium chloride and water (as a control), on qualitative and quantitative analysis of fifteen representative ginsenosides based on liquid chromatography hybrid quadrupole-time of flight mass spectrometry (LC–Q-TOF/MS). To evaluate the influence of mobile phase additives on qualitative performance, the quality of the negative mode MS/MS spectra of ginsenosides produced by online LC–Q-TOF/MS analyses, particularly the numbers and intensities of fragment ions, were compared under different adduct ion states, and found to be strongly affected by the mobile phase additives. When 0.02% acetic acid was added in the mobile phase, the deprotonated ginsenosides ions produced the most abundant product ions, while almost no product ion was observed for the chlorinated ginsenoside ions when 0.1mM ammonium chloride was used as the mobile phase additive. On the other hand, sensitivity, linear range and precision were adopted to investigate the quantitative performance affected by different mobile phase additives. Validation results of the LC–Q-TOF/MS-based quantitative performance for ginsenosides showed that ammonium chloride not only provided the highest sensitivity for all the target analytes, but also dramatically improved the linear ranges, the intra-day and inter-day precisions comparing to the results obtained using other mobile phase additives. Importantly, the validated method, using 0.1mM ammonium chloride as the mobile phase additive, was successfully applied to the quantitative analysis of ginsenosides in rat plasma after intragastric administration of Ginsenoside Extract at 200mg/kg. In conclusion, 0.02% acetic acid was deemed to be the most suitable mobile phase additive for qualitative analysis of ginsenosides, and 0.1mM ammonium chloride in mobile phase could lead to the best quantitative performance. Our results reveal that choosing the appropriate mobile phase additive is an important step in optimizing the analytical conditions, and the best quantitative method may not be suitable for the qualitative analysis.

Determination of Chloramphenicol in Honey, Shrimp, and Poultry Meat with Liquid Chromatography–Mass Spectrometry: Validation of the Method According to Commission Decision 2002/657/EC

Chloramphenicol (CAP) is an antibiotic used for the treatment of bacterial infections in human and veterinary medicine. The use of CAP was prohibited in the European Union in 1994. Control laboratories are required to use suitably validated analytical methods to check sample compliance with the regulation. A quantitative method based on liquid chromatography coupled to isotopic dilution tandem mass spectrometry was developed for the determination of chloramphenicol in honey, shrimp, and poultry meat. The experimental protocol consisted of a liquid–liquid extraction with ethyl acetate. Separation and detection were realized, respectively, by a 2690 Waters HPLC (Milford, MA, USA) and a Micromass Triple Quadrupole mass spectrometer (Micromass, Manchester, UK), equipped with an electrospray source. The effects of mobile-phase additives on the response of LC/ESI/MS were examined. Two different HPLC columns were tested: the X-Terra from Waters and the Alltima HP C18 HL from Alltech (Deerfield, IL, USA). A validation of the method was conducted according to the EU criteria for the analysis of chloramphenicol in foods. The decision limits (CCα) were 0.04, 0.03, 0.07 μg kg−1, and the detection capabilities (CCβ) were 0.05, 0.04, 0.08 μg kg−1 for honey, shrimp, and poultry meat, respectively. Those values are below the minimum required performance limit set at 0.3 μg kg−1 by the EU and 0.1 μg kg−1 by Belgium. Our protocol has the advantage to propose a unique extraction method working as well for honey, shrimp, and poultry meat, contrary to similar published methods in which a different extraction method is used for each type of matrix.

Effect of Mobile Phase Additives on the Resolution of Four Bioactive Compounds by RP-HPLC

The use of mobile phase additives enhances the separation and resolution of the bioactive compounds on the C18 column. Chlorogenic acid, caffeic acid, rutin, and scoparone from Herba Artemisiae Scopariae were investigated as the target compounds. Acetic acid, triethylamine, inorganic salts, and several ionic liquids were added as mobile phase additives into methanol/water (40:60, v/v). The result revealed that a mobile phase with 0.01 mol/L of ionic liquid [BMIM][BF4] enabled the optimum separation of the four target compounds.

Effect of mobile phase additives on resolution of some nucleic compounds in high performance liquid chromatography

Here we investigate the chromatographic behavior, with reversed-phase high performance liquid chromatography (RP-HPLC) of nucleic compounds (nucleobases, nucleosides, and nucleotides) on a C18 column in several different mobile phase additives, including1-butyl-3-methylimidazolium tetrafuloroborate ([BMIm][BF4]), 1-ethyl-3-methylimidazolium methylsulfate ([EMIm][MS]) ionic liquids, ammonium formate, and potassium phosphate. The effect of the alkyl group length, the imidazolium ring, and the ionic liquid's counterions on retention and resolution of the samples were tested. The results show the potential application of a used buffer system, ion pairing system, and ionic liquid as mobile phase additives in liquid chromatography resolution of nucleic compounds.

Effect of Mobile-Phase Additives on Separation of Gold Nanoparticles by Size-Exclusion Chromatography

Two spherical gold nanoparticles (diameters 5.3 and 38 nm, respectively) were used to appraise the separation performance of various mobile-phase additives in size-exclusion chromatography (SEC). The three additives were sodium citrate (SC), sodium chloride (NaCl), and sodium dodecylsulfate (SDS). The problem of adsorption on gold nanoparticles occurs when SC and NaCl are used as mobile-phase additives. In contrast, the adsorption problem is overcome by using SDS as mobile-phase additive; furthermore, SDS prevents gold nanoparticles from coagulating during separation. This paper demonstrates the feasibility of using SDS as mobile-phase additive in SEC for size separation of gold nanoparticles and also demonstrates how SEC may be used to investigate interactions between a mobile-phase additive and gold nanoparticles.

Determination of aristolochic acids in medicinal plant and herbal product by liquid chromatography–electrospray–ion trap mass spectrometry

This paper describes a liquid chromatography–electrospray–ion trap mass spectrometry (LC–ES–ITMS) method for the determination of aristolochic acid I and II (AA-I and AA-II) in medicinal plants and Chinese herbal remedies. A reversed phase C 18 column with gradient elution was utilized. The effects of mobile phase additives, acetic acid and ammonium acetate, on LC separation and ES ionization were investigated. For both AA-I and AA-II, the [M+NH 4] + ion was found to be the precursor ion for target MS/MS analysis. The MS/MS product ion, [M+H−44] +, was used for the quantitative measurement of AA-I and AA-II. The linearity was good from 0.03 to 5 μg ml −1 and good correlation ( r 2=0.999) over the range examined was determined for both AA. The detection limit based on a signal-to-noise ratio of three was 0.012 and 0.015 μg ml −1 for AA-I and AA-II, respectively. Various Chinese herbal remedies obtained from renal failure patients and medicinal plants were examined by this newly developed method.

Liquid chromatographic resolution of racemic amines, amino alcohols and related compounds on a chiral crown ether stationary phase

A chiral stationary phase (CSP) based on diphenyl-substituted 1,1′-binaphthyl crown ether was applied in resolving various racemic amines, amino alcohols and α-aminocarbonyl compounds including pharmaceutically important compounds such as amphetamine analogues, mexiletine, norepinephrine and norephedrine. The resolution was quite successful. In order to find out the effects of mobile phase additives on the chromatographic resolution behaviors, four selected racemic compounds were resolved on the CSP with the variation of the type and content of organic, acidic and cationic modifiers in aqueous mobile phase and with the variation of column temperature. The resolution behaviors were quite dependent on the type and the content of organic, acidic and cationic modifiers in aqueous mobile phase and on column temperature.

Memory effect of mobile phase additives in chiral separations on a Chiralpak AD column

Using chiral probes shown to be sensitive to the presence of mobile phase additives, a memory effect for these additives by an amylosic column was demonstrated. Exposure to these additives gave prolonged chromatographic performance changes even after their removal from the mobile phase. This finding is consistent with strong binding of the additives to the stationary phase. A procedure to remove bound additives was developed.

Effects of mobile-phase additives, solution pH, ionization constant, and analyte concentration on the sensitivities and electrospray ionization mass spectra of nucleoside antiviral agents.

The effects of various mobile-phase additives, solution pH, pKa, and analyte concentration on electrospray ionization mass spectra of a series of purine and pyrimidine nucleoside antiviral agents were studied in both positive and negative ion models. The use of 1% acetic acid resulted in good HPLC separation and the greatest sensitivity for [M + H]+ ions. In the negative ion mode, 50 mM ammonium hydroxide gave the greatest sensitivity for [M - H]- ions. The sensitivities as [M + H]+ ions were significantly larger than the sensitivities as [M - H]- ions for purine antiviral agents. Vidarabine monophosphate and pyrimidine antiviral agents, however, showed comparable or greater sensitivities as [M - H]- ions. The sensitivity as [M + H]+ showed no systematic variation with pH; however, the sensitivity as [M - H]- did increase with increasing pH. At constant pH, the ion intensity of the protonated species increased with increasing pKa. At higher analyte concentrations, dimer (M2H+) and trimer (M3H+) ions were observed. [M + Na]+ adducts were the dominant ions with 0.5 mM sodium salts for these compounds. The spectra of the more basic purine antiviral agents showed no [M + NH4]+ adduct ions, but [M + NH4]+ ions were the major peaks in the spectra of the less basic pyrimidine antiviral agents with ammonium salts. The ammonium adduct ion was formed preferentially when the proton affinity of the analyte was close to that of NH3. Abundant [M + OAc]- ions were observed for all of the antiviral agents except vidarabine monophosphate from solutions with added HOAc, NaOAc, and NH4OAc. The utility of mobile phases containing 1% HOAc or 50 mM NH4OH was demonstrated for chromatographic separations.

Electrospray Ionization Mass Spectrometry of Tetracycline, Oxytetracycline, Chlorotetracycline, Minocycline, and Methacycline

The effects of mobile-phase additives and analyte concentration on electrospray ionization mass spectra of a series of tetracyclines were investigated in both positive and negative ion modes. Only [M + H](+) and [M - H](-) ions were observed. The greatest sensitivity as [M + H](+) ions was obtained with 1% acetic acid and the greatest sensitivity as [M - H](-) ions was obtained using 50 mM ammonium hydroxide. Sensitivities in the positive ion mode were greater than those in the negative ion mode. The sensitivity as [M + H](+) showed no systematic variation with pH; however, the sensitivity as [M - H](-) did increase with increasing pH. A larger linear range was observed for [M - H](-) than for [M + H](+) ions. Both [M + Na](+) and [M + H](+) ions were observed with 0.5 mM sodium acetate and sodium iodide, but no adduct ions were observed with ammonium acetate. Some M(2)H(+) ions were observed at higher concentrations. Cluster ions, Na(NaOAc)(n)(+) or Na(NaI)(n)(+), but no sample ions were observed using 5 mM salts. The data suggest that mechanisms in addition to solution ionization are involved in the formation of the ESI sample ions. The utility of mobile phases containing 1% HOAc or 50 mM NH(4)OH was demonstrated for chromatographic separations.

Study of the Effect of Mobile Phase Additives on Retention in Reversed Phase HPLC Using Linear Solvation Energy Relationships

Linear solvation energy relationships (LSERs) were used to delineate which specific intermolecular interactions are responsible for changes in retention for a variety of well characterized analytes when acidic and basic additives were used in reversed phase HPLC. The effects of trifluoroacetic acid, triethylamine and a combination of trifluoroacetic acid and triethylamine on the LSERs were compared to those observed in the absence of additives. These effects were examined using four different mobile phase modifiers and five different stationary phases. Trifluoroacetic acid alone and in combination with triethylamine produced LSER regression coefficients nearly identical to those obtained with no additive present in the mobile phase. Triethylamine alone produced different LSER regression coefficients from the other systems unless the mobile phase contained trifluoroethanol as the mobile phase modifier, or the stationary phase consisted of a polymeric support.

Effect of Mobile Phase Additives in Packed-Column Subcritical and Supercritical Fluid Chromatography

The effect of mobile phase additives is investigated for a variety of compounds under subcritical and supercritical conditions using packed columns. Retention of hydrogen bond donor/acceptor analytes was found to be more dependent on the presence of mobile phase additives than weak hydrogen bond acceptor analytes. The temperature and pressure of the mobile phase are major factors in the extent of this dependence. Consequently, selectivity between homologous compounds is dependent on both the additive used and the state of the mobile phase. Efficiency is nearly always improved by the presence of mobile phase additives, more so under supercritical conditions than under subcritical conditions. These observations suggest that surface molar excesses of mobile phase additives play a large part in the resulting character of the supercritical chromatographic system.

Effect of mobile phase additives on peptide retention in reversed-phase chromatography with pellicular and totally porous sorbents

The effect of two mobile phase additives, trifluoroacetic acid and phosphoric acid, on the energetics of peptide retention in reversed-phase chromatography was investigated using Hy-Tach C 18 micropellicular and Vydac C 4 and C 18 totally porous stationary phases. The effect of the relatively low phase ratio of columns packed with micropellicular sorbents was also examined. The logarithmic retention factors, of two model peptides, Ac-RGGGGLGLGK-amide and Ac-RGAGGLGLGK-amide, were evaluated with different columns and additives in a practical range of eluent strength. The dependence of the logarithmic retention factor on the concentration of acetonitrile in the mobile phase was linear in all cases. The higher sensitivity of the retention to the organic modifier concentration in the case of the Hy-Tach C 18 column is attributed to the relatively low phase ratio of this column. Pairwise plots of the logarithmic retention factors were linear. The plots of data obtained with the two additives has unit slopes and thus reveal homoenergetic retention behavior. On the other hand data obtained on two different columns manifest homooenergetic retention, the slopes of plots are different from unity. The analysis has yielded consistent results and validated the assumption that the retention free energy can be divided into two components arising from mobile phase and stationary phase contributions. The approach also allowed an estimation of the relative phase ratios of the columns and the Vydac C 18 column was found to have an 3 and 8 times higher phase ratio than the Vydac C 4 and the Hy-Tach C 18 column, respectively.

Determination of beta-lactam residues in milk using perfusive-particle liquid chromatography combined with ultrasonic nebulization electrospray mass spectrometry.

The introduced electrospray (ESP) technique combined with quadrupole mass spectrometry (MS) was applied for the trace residue detection (10 ppb) of commonly administered beta-lactam antibiotics in bovine milk. Because of the widespread use of penicillin G, ampicillin, amoxicillin, cephapirin, cloxacillin, and ceftiofur in veterinary medicine, these six popular drugs were chosen to develop a fast and reliable microcolumn liquid chromatographic (micro-LC) separation method for residue-containing milk extracts. Furthermore, the analytes were selected to test the chromatographic behavior of a novel stationary phase, a perfusive-particle column packed with derivatized porous polystyrene divinylbenzene. The effects of mobile phase additives on separation and ESP ionization efficiency were investigated. The ionization of the eluted analytes took place either in a conventional ESP interface with a capillary shield allowing flow rates up to 40 microL/min or in the latest model of the ultrasonic nebulization ESP interface. The perfusive-particle column, although not optimized for the separation of small molecules, exhibited sufficient resolution of the antibiotics for its routine usage, especially when considering the remarkable time-saving advantage compared with ordinary reversed phase micro-LC columns. The application of the novel ultrasonic nebulization interface lowered detection limits and improved the stability of the signals over a much wider flow rate range than possible for ionization with the ESP system equipped with the conventional needle.