Nitrile Column Research Articles

LC–MS Determination of Gabapentin from Human Plasma

Gabapentin is an anticonvulsant drug used for the treatment of epilepsy. It is not bound to plasma protein and is not metabolized. A high performance liquid chromatography–mass spectrometric micro method is described in this report for its determination from human plasma. Chromatography was performed on a 50 × 4.6 mm, 4 μm nitrile column and the parent ion detected in the positive ionization mode on single quadrupole analyzer (Q1MI) with atmospheric pressure ionization source. Extraction was carried out on C18, 100 mg/3cc cartridge using 10 μL sample volume. The mean extraction recovery was 97% and within batch and between batch coefficients of variation were <9%. Lack of interference from endogenous substances helped in achieving a highly sensitive method without the need for monitoring fragment ions. The lowest concentration injected on column for calibration curve was 195 pg (range 0.5–64 ng). The method was applied for analysis of samples from a cross-over bio-equivalence study comparing two formulations.

A novel HPLC method reveals that precipitation of 2′-deoxyadenosine 5′-monophosphate with lithium perchlorate/acetone leads to base depurination

A novel HPLC method reveals that precipitation of 2′-deoxyadenosine 5′-monophosphate with lithium perchlorate/acetone leads to base depurination

Gradient elution in normal-phase high-performance liquid chromatographic systems

Gradient elution is widely used for separation of complex samples in reversed-phase HPLC systems, but is less frequently applied in normal-phase HPLC, where it has a notoriously bad reputation for poor reproducibility and unpredictable retention. This behaviour is caused by preferential adsorption of polar solvents used in mixed mobile phases, which may cause significant deviations of the actual gradient profile from the pre-set program. Another important source of irreproducible retention behaviour is gradual deactivation of the adsorbent by adsorption of even traces of water during normal-phase gradient elution. To avoid this phenomenon, carefully dried solvents should be used. Finally, column temperature should be carefully controlled during normal-phase gradient elution if reproducible results are to be obtained. Working with dry solvents at a controlled constant temperature and using a sophisticated gradient-elution chromatograph, reproducibility of the retention data in normal-phase gradient elution better than 2% may be achieved even over several months of column use. The retention data in gradient elution can be calculated accurately if appropriate corrections are adopted for the gradient dwell volume and for the preferential adsorption of the polar solvents using experimental adsorption isotherms. The average error of prediction for the corrected calculated gradient retention data was lower than 2% for a silica gel column and lower than 3% for a bonded nitrile column, which may be suitable for the optimization of separation. Further, a simple approach is suggested for rapid estimation of changes in the retention induced by a change in the gradient profile in normal-phase HPLC. For such a rough estimation, it is not necessary to know the parameters of the dependence of the solute retention factors on the composition of the mobile phase.

Simultaneous determination of cinnarizine and domepiridone maleate from tablet dosage form by reverse phase ion pair high performance liquid chromatography

A new simple, precise, rapid and selective reverse phase ion pair high performance liquid chromatography (HPLC-RP) method has been developed for the simultaneous determination of cinnarizine (CINN) and domepiridone maleate (DOME) from tablets using acetonitrile–methanol–water–0.1 N sulfuric acid (37:10:48:5 v/v/v/v) containing sodium lauryl sulfate (0.01 M), as a mobile phase and a Machery Nagel nitrile column (10 μ, 25 cm×4.0 mm i.d.) as the stationary phase. The flow of mobile phase through the column was kept at 1.0 ml min −1 through out the analysis. Detection was carried out using a UV detector at 225 nm. The retention times for CINN and DOME were 4.73 and 9.41 min, respectively. The linearity range and percentage recoveries for CINN and DOME were 4–1000 and 60–750 μg ml −1 and 99.90 and 99.60%, respectively.

Optimisation of gradient elution in normal-phase high-performance liquid chromatography

An optimisation approach for linear gradient elution in normal-phase chromatography was suggested. The approach is based on predictive calculations of the retention and of the resolution of the individual pairs of compounds in the sample mixture. Isocratic or gradient-elution retention data, acquired in a few initial runs under different conditions, are employed to determine the parameters of retention equations describing the dependence of the retention factor, k, on the composition of the mobile phase. Unlike earlier procedures, a more complex, three-parameter retention equation can be used as the basis of predictive calculations, if necessary. The approach allows one to use either maximised minimum resolution in the sample mixture or the minimum time necessary to achieve the required resolution as the optimisation criterion. It accounts for the contribution of the initial isocratic elution step induced by the gradient dwell volume, so that it is not necessary to delay the injection with respect to the start of the gradient. Simultaneous optimisation of the gradient slope and of the gradient range is performed, with the gradient volume (time), plate number and other variables as adjustable parameters. The approach is illustrated by examples of gradient-elution separation of phenylurea herbicides on a silica gel and on a bonded nitrile column, with binary gradients of 2-propanol in n-heptane and of dioxane in n-heptane. Dried solvents and the temperature (controlled to ±0.1°C) were used to improve the reproducibility of the retention data.

Determination of molybdenum in various materials by normal-phase liquid chromatography using N-benzoyl- N-phenylhydroxylamine

A normal-phase liquid Chromatographie method for the selective determination of molybdenum with N-benzoyl- N-phenylhydroxylamine is described. The molybdenum(VI) complex was preconcentrated by extraction into chloroform and injected onto a nitrile column for chromatography. The mobile phase was a 0.075 M solution of reagent in chloroform (stabilized with amylene). The detection limit for molybdenum by the proposed method was 0.88 ng for a phase volume ratio of 20:1 (aqueous to organic). Molybdenum has been determined in several samples with satisfactory accuracy and precision.

Lutein associated with a transthyretin indicates carotenoid derivation and novel multiplicity of transthyretin ligands

Transthyretins isolated from different species bind hydrophobic compounds and are often obtained in a yellow form. Such a transthyretin from chicken serum was purified by chromatography using Sepharose-coupled human retinol-binding protein. The yellow chromophore was extracted with methanol and purified by reverse phase HPLC followed by normal-phase chromatography on a nitrile column. Ultraviolet-visible absorbance and mass spectrometry identified the yellow compound as lutein, i.e. xanthophyll, ( all-trans)-β,ϵ-carotene-3,3′-diol, estimated to constitute 10–30% of associated colourless compounds. These components are different from the yellow component isolated from human transthyretin and establish that carotenoid-derived pigments can be associated with transthyretins.

Liquid Chromatographic Methods for Fluoxetine Hydrochloride, Its meta Isomer, and Related Compounds in Raw Materials

Abstract Liquid chromatographic (LC) methods were developed for assay of fluoxetine hydrochloride, its meta isomer, and 12 related compounds in drug raw materials. The method for assay and determination of the meta isomer requires an Exsil ODS-B column; a mobile phase consisting of tetrahydrofuran, acetonitrile, and buffer; a flow rate of 1 mL/min; and detection at 214 nm. The method for related compounds specifies a Spherisorb nitrile column; a mobile phase consisting of tetrahydrofuran, acetonitrile, and buffer; a flow rate of 1 mL/min; and detection at 214 nm. The limits of quantitation in the presence of the drug are 0.1% for the meta isomer and 0.05% or less for the related compounds. Assay precision was less than 1.0% on triplicate weighings. Drug contents of 4 raw material samples were between 98.8 and 100.1%. Impurity levels in the 4 samples ranged from 0.14 to 0.45%. In addition, one sample contained ca 0.13% of the meta isomer. A second analyst, using columns from different suppliers, obtained similar results.

Liquid Chromatographic Determination of Amiodarone Hydrochloride and Related Compounds in Raw Materials and Tablets

Abstract A liquid chromatographic method for the determination of amiodarone hydrochloride and 10 related compounds in drug raw material and for assay of drug in tablets was developed. The method specifies a 3 jxm Hypersil nitrile column (150 × 4.6 mm), a mobile phase of 1 + 1 acetonitrile–ammonium acetate buffer (0.1 M adjusted to pH 6.0 with 0.1 M acetic acid), a flow rate of 1 mL/min, and detection at 240 nm. The lower limit of quantitation of the related compounds is 0.02% or less. Drug contents in 2 raw material samples were 100.1 and 99.9% and ranged from 98.2 to 99.4% in 3 tablet formulations. Impurity levels in 2 samples of raw material from different manufacturers were ca 0.4%. The presence of 3 of the known related compounds in these samples was confirmed by liquid chromatographymass spectrometry. The method applied to raw materials was evaluated by a second laboratory and found to be satisfactory.

Determination of glutaraldehyde in solution as its bis-2,4-dinitrophenylhydrazone derivative; determination of geometrical isomer ratios

A reversed-phase HPLC method for the determination of glutaraldehyde in fresh 2% w/v activated alkaline solutions was developed, which used derivatization with 2,4-dinitrophenylhydrazine to its bis-2,4-dinitrophenylhydrazone. The chromatographic conditions were based on those for the Occupational Safety and Health Administration method for the determination of glutaraldehyde in air. The column was a 250 × 4.5 mm Zorbax 5 μm nitrile column, with a mobile phase of acetonitrile: 0.1% v/v aqueous phosphoric acid (62:38) at a flow rate of 1 ml/min. The injection volume was 10 μl and UV detection was at 365 nm. Derivatization was maximal after 80 min. The chromatography was linear in the range 0.02–0.2 μg glutaraldehyde injected and the whole method was linear in the range 0.8–4% w/v glutaraldehyde. The precision of chromatography was in the range 0.3–0.5% relative standard deviation and the within-run precision was ±0.035% w/v (95% confidence limits). Derivatized samples and standards were stable for at least three months in the dark at room temperature: the stability data were used to estimate the day-to-day precision as ±0.09% w/v (95% confidence limits). The mean recovery of glutaraldehyde spiked into Cidex brand glutaraldehyde solution was 97.3%±3.2% (95% confidence limits) and to Asep was 101.8%±3.4% (95% confidence limits). The E,E and E,Z geometric isomers of glutaraldehyde-bis-2,4-dinitrophenylhydrazone were formed in the same proportions in both samples and standards. However, with the isolated bis-hydrazone, the proportions were different, making it potentially unsuitable as a standard as an alternative to glutaraldehyde solution.

Chiral mobile phases in the enantiomeric analysis and in the evaluation of stability constants of π-π complexes

Abstract

High-performance liquid chromatographic assay of debrisoquine and its 4-hydroxy metabolite in human urine

An isocratic HPLC method is described. It allows the simultaneous determination of debrisoquine and 4-hydroxydebrisoquine in urine. Sample preparation involves a rapid and simple (one step) liquid-liquid extraction without derivatization. Separation, followed by UV-detection, is carried out on a nitrile column in reversephase mode with a binary, aqueous mobile phase. This rapid assay uses a new internal standard (guabenxane) and allows the determination of urinary concentrations with good precision and appropriate sensitivity.

Simultaneous Determination of Minoxidil and Tretinoin in Pharmaceutical and Cosmetic Formulations by Reversed-Phase HPLC

Abstract A method based on reversed-phase HPLC on a nitrile column, has been developed for the simultaneous determination of minoxidil and tretinoin. Detection limits were 10 ng of minoxidil and 5 ng of tretinoin injected, at 283 and 367 nm, respectively. Linearity was observed up to 5 μ. The method has been applied for the analysis of commercial cosmetic and pharmaceutical formulations.

Liquid Chromatographic Determination of Pindolol and Related Compounds in Raw Materials

A liquid chromatographic method for the assay of pindolol and related compounds in the bulk drug has been developed. The method resolves 6 known and several unknown impurities from the drug and each other by using a nitrile column, an acetonitrile-sodium acetate buffer (35 + 65), and a UV detector set at 219 nm. Minimum quantifiable amounts of impurities are 0.02% or less relative to the drug. Ten lots of pindolol raw material were evaluated for purity and drug content. Total levels of impurities in these samples, quantitated against pindolol, ranged from about 0.03 to 0.24%. Assay results were within the range of 98.5 to 101.5%.

Determination of B6Vitamers in Serum by Simple Isocratic High Performance Liquid Chromatography

Abstract A high-performance liquid chromatographic (HPLC) method for the determination of individual B6 vitamers was developed, and its application to dog, rat, monkey and human serum is described. Serum samples were deproteinated with 8% aqueous perchloric acid and the extracts were purified using an anion-exchange column. Analytical separation of the B6 vitamers and the internal standard (4′-deoxypyridoxine) was achieved by isocratic elution with a nitrile phase column in less than 30 minutes. The described methodology is simple and fast, and yields high percentage recoveries (> 90) of the vitamers with low inter- and intra-day variations.

Determination of 2-methyl derivatives of tamoxifen in cell culture medium using high-performance liquid chromatography and electrochemical detection.

The 2-methyl derivatives of tamoxifen (2-methyltamoxifen and 2-methyl-4-hydroxytamoxifen) were extracted from a cell culture medium at pH 5.4 (Earle's Minimum Essential Medium) with an internal standard (tamoxifen) on a phenyl sorbent cartridge. The compounds were then separated by high-performance liquid chromatography on a nitrile column eluted with acetonitrile-methanol-0.05 M sodium dihydrogenphosphate (19:4:11.6:69,v/v) containing 0.11 mmol/l disodium EDTA and determined by electrochemical detection at +1.1 V vs. Ag/AgCl/3 M NaCl. The absolute detection limits were 50 pg for 2-methyl-4-hydroxytamoxifen and 100 pg for tamoxifen and 2-methyltamoxifen at a sensitivity of 1 nA/V.

Sensitive and specific high-performance liquid chromatographic assay for the quantification of sulforidazine and two diastereomeric sulforidazine-5-sulfoxide metabolites in plasma

A sensitive and specific procedure using high-performance liquid chromatography for the quantification of sulforidazine and two diastereomeric sulforidazine-5-sulfoxide metabolites in plasma was developed. Sulforidazine was first extracted from basified plasma using a mixture of pentane and 2-propanol. Sulforidazine-5-sulfoxide metabolites were then extracted from the same basified plasma using a second solvent mixture consisting of methylene chloride, pentane and 2-propanol. Each organic extract was subsequently back-extracted separately with 0.1 M hydrochloric acid, basified and re-extracted with the original solvent mixtures. In order to avoid interferences due to hydroxylated metabolites co-eluting with sulforidazine-5-sulfoxides in the more polar extract, this extract was derivatized with N-methyl-( tert.-butyldimethylsilyl)trifluoroacetamide. The two extracts were separately chromatographed on a narrow-bore nitrile column using ultraviolet detection. The quantification limits for sulforidazine and two diastereomeric sulforidazine-5-sulfoxide metabolites were 1.0 ng/ml with mean intra-assay coefficients of variation less than 10%. These methods were applied to the analysis of plasma from a dog following the administration of a single oral dose (25 mg of the base) of sulforidazine hydrochloride.

Chromatographic separation and quantitative determination of the metabolites of di-(2-ethylhexyl) phthalate from urine of laboratory animals

Free, glycine-conjugated, and glucuronide-conjugated metabolites of di-(2-ethylhexyl) phthalate may be stripped from urine with XAD-2 resin, derivatized, and quantitatively analyzed by liquid chromatography on a nitrile column with UV and/or radioactivity monitors. One class of metabolites requires reversed-phase chromatography or gas—liquid chromatography for its resolution. Relative molar responses of the hydrogen flame-ionization detector to these metabolites have been determined. Packed gas chromatography columns (OV-3, OV-210, cyclohexanedimethyanol succinate) and fused-silica capillary columns (SP2100 and FFAP) are useful for quantitative analysis under appropriate conditions. The simplest gas chromatographic procedure permitting complete quantitative analysis requires hydrolysis of conjugates, formation of methyl esters of carboxyl groups, butyration of hydroxyl groups and chromatography on OV-3. Typical distributions of di-(2-ethylhexyl) phthalate metabolites in urine from mice, hamsters and guinea pigs are presented.

Gas Chromatography of Phenylphenols and Chlorophenylphenols on a Silicone Nitrile Column

Gas Chromatography of Phenylphenols and Chlorophenylphenols on a Silicone Nitrile Column