Volatile Ion-pairing Agent Research Articles

Quantitation of levodopa and carbidopa in rat plasma by LC-MS/MS: The key role of ion-pairing reversed-phase chromatography.

A simple and selective bioanalytical method was developed for simultaneous determination of levodopa and carbidopa in rat plasma by LC-MS/MS. Levodopa and carbidopa are small polar molecules, posing challenges in the development of selective and efficient chromatography conditions. Perfluoropentanoic acid (PFPA), a volatile ion-pairing agent, was utilized to enhance chromatographic characteristics of both compounds in the reversed-phase mechanism. The ion-pairing chromatography played an essential role in mitigating matrix effects and achieving adequate separation between interfering background peaks and those of the analytes of interest, especially for levodopa. A 96-well based, automated liquid-liquid extraction, via the use Hamilton NIMBUS liquid handlers, was developed. Butyl alcohol, when mixed with ethyl acetate, greatly increased the recovery of both levodopa and carbidopa. The addition of PFPA further enhanced recovery for both analytes. Sodium metabisulfite, an antioxidant, was used to stabilize levodopa and carbidopa in rat plasma. The method was validated in the ranges of 50-10,000ng/mL and 25-5000ng/mL for levodopa and carbidopa, respectively, using levodopa-d3 and carbidopa-d3 as internal standards. The validated method was successfully applied to analyze rat plasma samples from in-life studies.

Improved liquid chromatographic method with pulsed electrochemical detection for the analysis of kanamycin

This work describes the separation of the main component kanamycin A from its related substances using an improved liquid chromatographic method with pulsed electrochemical detection (LC-PED). Two methods, one using volatile ion pairing agents and the other using non-volatile ones were developed. Using volatile additives, the total run time was rather long with no possibility of developing gradient elution. The non-volatile method was found to be more performant and hence was selected for further quantitative work. This method employed gradient elution in order to reduce the analysis time and to improve the sensitivity of the late eluting peaks. Mobile phase A consisted of sodium sulphate (5.0 g/l), sodium octanesulphonate (0.5 g/l) and 0.2 M phosphate buffer pH 3.0 (50.0 ml/l). Mobile phase B was the same as A except for the amount of sodium sulphate which was increased to 15 g/l. Using a Platinum EPS column (150 mm × 4.6 mm ID, 3 μm) kept at 45 °C, 22 components could be separated within 45 min indicating that this method is much more selective than other already published ones. Robustness of the method was examined by means of an experimental design. The limit of detection and limit of quantitation were found to be 1.7 and 5 ng, respectively. The method was found to be linear in the range LOQ–600 ng injected with a coefficient of determination equal to 0.999.

Quantitative determination of glycopyrrolate in human plasma by liquid chromatography–electrospray ionization mass spectrometry: The use of a volatile ion-pairing agent during both liquid–liquid extraction and liquid chromatography

The work presented here deals with the development of a quantitative tool for the determination of the quaternary ammonium anticholinergic glycopyrrolate in human plasma samples. Mepenzolate was used as an internal standard. The plasma samples were subjected to a suitable sample clean-up consisting of a simple and relatively fast, two step liquid–liquid ion-pair extraction procedure. The chromatography, using the same volatile ion-pair reagent heptafluorobutyric acid (HFBA), takes only 10 min. Relative standard deviation of retention times was never above 2.26% ( n = 36). The method was fully validated based on the US FDA Bioanalytical Method Validation Guidance for Industry. As such, a quantitative ESI-LC–MS(/MS) (TOF mass spectrometry) method was optimized for the absolute quantification of glycopyrrolate in human plasma in a concentration range from 0.101 to 101 ng/mL using a quadratic calibration function ( R 2 = 0.9995), y = −2.21 × 10 −4 (±3.93 × 10 −5) × x 2 + 5.85 × 10 −2 (±5.27 × 10 −3) × x + 4.08 × 10 −3 (±4.82 × 10 −4). For the three QC concentrations (QC 1 0.252, QC 2 2.52, and QC 3 25.2 ng/mL) and the LLOQ (0.101 ng/mL), total precision was under 20% (18.0% ( n = 6) at the LLOQ) and maximum accuracy was 112% (88.9% for the LLOQ, n = 6). Absolute matrix effect (maximum 133% ± 9.59, n = 3), absolute recovery (better than 41.8% ± 2.22, n = 3), relative (inter-subject) matrix effect (maximum 10.9% ± 1.45, n = 4) and process efficiency (better than 45.2% ± 5.74, n = 3) too were assessed at the 3 QC concentrations.

Characterization of impurities in sisomicin and netilmicin by liquid chromatography/mass spectrometry

The characterization of unknown impurities present in netilmicin and sisomicin by liquid chromatography (LC) coupled with mass spectrometry (MS) is described. The volatile ion-pairing agent trifluoroacetic acid (TFA) was used for the retention of the main compounds and their impurities on a reversed-phase (RP) C18 column, because they are highly hydrophilic and basic compounds. The method showed good separation between netilmicin and its four potential related substances prescribed in the European Pharmacopoeia, which were identified by comparison of their retention times with those of the reference substances. Furthermore, in total 16 unknown impurities in a netilmicin sample and six in a sisomicin sample with unknown identity were detected. The structures of the unknown compounds were deduced based on comparison of fragmentation patterns with those of the reference substances investigated in LC/MSn experiments by the use of electrospray ion trap mass spectrometry.

Improved liquid chromatographic method with pulsed electrochemical detection for the analysis of gentamicin

The official method for the determination of the composition and related substances of gentamicin prescribed by the European Pharmacopoeia (Ph. Eur.) is liquid chromatography combined with pulsed electrochemical detection (LC-PED). However, this method utilizes a polymer stationary phase which shows rather low efficiency towards the separation of the main gentamicin components. Moreover, the mobile phase contains a lot of non volatile salts: sodium sulphate and sodium octanesulphonate. Following a comparative study, the most performant LC–PED method has been evaluated and validated using a reversed phase C18 column (C18, 250 × 4.6 mm ID, 110 Å, 5 μm) kept at 35 °C with a mobile phase containing volatile ion pairing agents: trifluoroacetic acetic acid (TFA) and pentafluoropropionic acid (PFPA). In addition to the selectivity of the main gentamicin components and its related substances, the method is repeatable, linear and proves to be robust. It is also applicable to a wider number of C18 columns.

Rapid and sensitive determination of ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid in water samples by ion-pair reversed-phase liquid chromatography–electrospray tandem mass spectrometry

A new method is presented for the quantitative determination of ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA) from aqueous samples without an enrichment step. It consist of the formation of the Fe(III) complexes of EDTA and DTPA, liquid-chromatography with a volatile ion-pairing agent and determination by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Limits of quantification (LOQ) of 1.0 and 0.6 μg L −1 for EDTA and DTPA were obtained, allowing the direct injection of most aqueous environmental samples without any preceeding enrichment. With a more recent mass spectrometer, the LOQ could be further decreased by almost one order of magnitude. Parallel analysis of real samples by a standardized method employing enrichment, derivatization and GC–MS analysis yielded comparable results. The method was applied to the determination of both complexing agents in several wastewater, surface water and drinking water samples, showing that EDTA is an omnipresent contaminant in partially closed water cycles.

Effects of functional groups on the fragmentation of dyes in electrospray and atmospheric pressure chemical ionization mass spectra

Electrospray ionization (ESI) is most suitable for dyes with an ionic or very polar character, while atmospheric pressure chemical ionization (APCI) suits better those dyes and pigments with low to medium polarity. The negative-ion mode of ESI is used for anionic dyes such as (poly)sulfonated and/or (poly)sulfated variants, where the series of deprotonated molecules enables an easy determination of molecular weights and the number of acid groups. The positive-ion APCI or ESI mass spectra of non-ionic and cationic dyes and pigments provide typical molecular adducts [M + H] +, [M + Na] + and [M + K] + used for the molecular assignment. The first-order mass spectra often suffer from the lack of fragment ions, which can be solved by using multistage tandem mass spectra (MS n ) measured with an ion trap analyzer. The effects of various functional groups on the fragmentation behavior are described herein. The detailed understanding of fragmentation paths enables the correlation among particular functional groups and the fragmentation patterns. The presented approach is applicable for the structural analysis of complex mixtures using HPLC/MS/MS techniques with volatile ion-pairing agents.

Simple analysis of four bisphosphonates simultaneously by reverse phase liquid chromatography using n-amylamine as volatile ion-pairing agent

Volatile organic amine was used as the mobile phase addictive during the separation of four bisphosphonates (alendronate, pamidronate, zoledronic acid and etidronate). An isocratic liquid chromatography method with evaporative light-scattering detection (ELSD) was developed for these bisphosphonates which are not retained on non-polar column and lack chromophore for detection. The analytes have sufficiently separated from each other on a Phenomenex C 18 column. The effects of mobile phase composition and instrumental parameters of ELSD were studied. This newly developed method enables direct measurement for analysis of bisphosphonates without the need of derivatization. This developed method provides high separation and specificity to bisphosphonate analysis. In quantitative analysis, the method showed satisfactory precision (less than 2.8%) and accuracy (higher than 94.4%), good linearity ( r = 0.9991–0.9997) and sufficient sensitivity (15–18 μg/ml). It can be easily and conveniently adopted for the routine quality control analysis.

Sulfobutyl ether-β-cyclodextrin fingerprint using ion pair reversed-phase chromatography

A liquid chromatography (LC) method using volatile ion-pairing agent and evaporative light scattering detection has been developed for the analysis of sulfobutyl-ether-β-cyclodextrins (SBE-β-CD). Different protonated aliphatic amines (triethylamine, pentylamine and heptylamine) were evaluated as volatile ion-pairing agents. The effects on SBE-β-CD retention of the n-alkyl chain length and concentration (0.5 to 50 mM) of the ion-pairing agent and the effect of organic modifier (methanol or acetonitrile) addition in mobile phase were examined.

Use of volatile amines as ion-pairing agents for the high-performance liquid chromatographic–tandem mass spectrometric determination of aromatic sulfonates in industrial wastewater

The use of trialkylamines (triethylamine, N,N-dimethyl-n-butylamine, and tri-n-butylamine) as volatile ion-pairing agents for the high-performance liquid chromatographic separation of aromatic sulfonates was investigated. Negative ion electrospray tandem mass spectrometry was used to detect both singly and multiply charged analyte ions. Sensitivity of detection was strongly reduced by amine concentrations above 2.5 mmoll−1 in the eluent. With tributylamine as ion-pairing agent 19 aromatic sulfonic acids could be determined using time-scheduled selected reaction monitoring. Lowest orders of detection range from 3 to 74 μgl−1. Several naphthalenesulfonic acids were determined in dyeing baths and textile wastewater in concentrations ranging from 0.1 mgl−1 to 2.1 mgl−1. A degradation product of sulfonated azodyes was identified in the effluent of a laboratory treatment plant.

Ion-pair reversed-phase liquid chromatography for determination of polar underivatized amino acids using perfluorinated carboxylic acids as ion pairing agent

An isocratic liquid chromatography method using volatile ion pairing agent and evaporative light scattering detection was developed for the analysis of 10 underivatized amino acids (Asp, Asn, Ser, Gly, Gln, Cys, Glu, Thr, Ala, Pro) which are not sufficiently retained and resolved in the LC system commonly used. Five perfluorinated carboxylic acids (trifluoroacetic, heptafluorobutyric, nonafluoropentanoic, tridecafluoroheptanoic and pentadecafluorooctanoic acid) have been evaluated as ion-pairing agents in various concentrations (0.25–1 m M) using three analytical reversed-phase columns (LiChrospher RP-Select B, Purospher RP-18e and Supelcosil ABZ + Plus). The quantities adsorbed on the chromatographic supports and the equilibration times of each system are also studied. Excellent separation with satisfactory baseline resolution was achieved on the chromatographic system made up of a Purospher RP-18e and an aqueous mobile phase containing pentadecafluorooctanoic acid at 0.5 m M.

HYDROPHILIC ANIONS ANALYSIS USING REVERSED PHASE CHROMATOGRAPHY COUPLED WITH EVAPORATIVE LIGHT-SCATTERING DETECTION

A Liquid Chromatography (LC) method using volatile ion pairing agents and Evaporative Light Scattering Detector (ELSD) was developed. The separation of a series of three common inorganic anions (Cl−, NO3 − and SO4 2−) and three organo–phosphonic anions (Methyl, ethyl and propylphosphonic acids) was achieved on a reversed–phase C18 column with pentylammonium or heptylammonium formiate as ion pairing agent. This method provides good linearity of the calibration curve and enables a limit of detection to be reached of 4 mg/L and 3.12 mg/L for alkylphosphonic anions and inorganic anions respectively. The separation for methyl and ethylphosphonic acids, which are poorly resolved in the LC systems commonly used, was successfully achieved.

Volatile ion-pairing agents for liquid chromatographic—thermospray mass spectrometric determination of amino acids and amino acid amides

The behaviour of volatile ion-pairing agents in terms of performance of the combined liquid chromatography—mass spectrometry is described. The applicability of heptafluorobutanoic acid, tridecafluoroheptanoic acid and nonadecafluorodecanoic acid as volatile ion-pairing agents is demonstrated by the analysis of mixtures of amino acids and the corresponding amino acid amides. Translation of a non-volatile to a volatile ion-pair system that is suitable for thermospray mass spectrometry is described. Using nonadecafluorodecanoic acid, the retention times of the compounds studied were comparable to those obtained with n-dodecylsulphonic acid. Further, with nonadecafluorodecanoic acid, the repeatability of the tests and the calibration graphs of the compounds investigated were good. The sensitivity of the mass spectrometer towards the compounds of interest was greatly improved by postcolumn addition of trifluoroacetic acid and ionization with gaseous ammonia. Using nonadecafluorodecanoic acid, the method was applied to the analysis of samples from bio-organic synthesis.

Determination of aminoglycoside antibiotics by reversed-phase ion-pair high-performance liquid chromatography coupled with pulsed amperometry and ion spray mass spectrometry

This work constitutes a preliminary investigation of a high-performance liquid chromatographic (HPLC)-mass spectrometric (MS) method for confirming aminoglycoside residues in bovine tissues. A reversed-phase ion-pair HPLC method for the separation of four aminoglycosides was developed using volatile ion-pairing agents and optimized for detection with an ion spray HPLC-MS interface. The method is also compatible with a commercial pulsed amperometric detector that was used for HPLC method development and that may be useful for the screening and quantification phases of a regulatory method. Several column phases, eluent compositions, and pairing ions were evaluated for optimum HPLC-MS sensitivity. Detection limits are in the low nanogram range with the pulsed amperometric detector and with HPLC-MS in the selected ion monitoring mode. Results with bovine kidney, fortified to 20 ppm and extracted by matrix solid-phase dispersion, obtained using both detectors are presented.