Sensitive LC Method Research Articles

LC determination of leuprolide component amino acids in injectable solution by phanquinone pre-column derivatization labelling procedure

A sensitive LC method for the determination of leuprolide acetate component amino acids in injectable solution with fluorogenic pre-column derivatization has been developed. The derivatization reaction with phanquinone was optimised by a series of experiments. Histidine, arginine, serine, tryptophan, glutamic acid, tyrosine, methionine, isoleucine, leucine and phenylalanine were separated on a reversed-phase ODS column using as eluent a binary mixture of triethylammonium phosphate buffer–methanol, under gradient elution conditions. The derivatives were eluted in 30 min with good reproducibility. The hydrolysis reaction of the peptide was carried out at reflux with 12N hydrochloric acid for 2 h 30 min. The intra-day accuracy of the entire procedure (hydrolysis, derivatization, LC separation) ranged from 80.5 to 109.5% of the nominal concentration of leuprolide acetate and the precision (%R.S.D.) was less than 5.8%; the inter-day accuracy was in the range 81.5–107.2% and corresponding R.S.D. values were less than 4.6%. The detection limits (signal-to-noise ratio = 3) for the adducts are 30–800 fmol.

A simple HPLC method with pulsed EC detection for the analysis of creatine

The objective of this study was to develop a simple and sensitive LC method for the determination of creatine in aqueous solutions as well as in rat plasma using electrochemical detection. The chromatographic system consisted of a GP50 gradient pump, an ED40 pulsed electrochemical detector, and an AI-450 chromatography automation system (Dionex). The mobile phase consisted of a mixture of water, acetonitrile, 0.01 M sodium acetate, and 1.0 M sodium hydroxide (2.5:2.5:90:5, V/V/V/V) at a flow rate of 1.0 ml/min. The chromatographic separation was achieved at 45 °C on a column with a polyhydroxylated glucose and sulfonated stationary phase. The retention times of creatine and creatinine was 3.50 and 4.73 min, respectively, with creatine fully resolved from its major degradation product, creatinine. The standard curves were linear over the concentration range of 0–20 μg/ml. Within-day and day-to-day relative standard deviations (R.S.D.) were less than 10%. This method was used to study dissolution characteristics of various creatine salts in water.

Liquid chromatography analysis of enrofloxacin and ciprofloxacin in chicken blood spotted on filter-paper disks

A simple, low-cost, sensitive and selective LC method was developed for the determination of enrofloxacin and ciprofloxacin in chicken blood. The method was applied to whole blood from a chicken using dried blood spots on filter paper disks. The detection limits of enrofloxacin and ciprofloxacin (100 μl of whole blood on a disk) were 0.005 and 0.01 μg/ml, respectively. The whole procedure was verified in intra-laboratory studies (recoveries of both compounds were above 90%), and its applicability was tested with blood from the chicken receiving enrofloxacin in a single oral dose at a level of 10 mg/kg body mass. The method permits the use of a small volume of blood from a chicken and should be useful for pharmacokinetic studies.

A simple LC method with UV detection for the analysis of creatine and creatinine and its application to several creatine formulations

The objective of this study was to develop a simple and sensitive LC method for the determination of creatine and creatinine in various creatine supplement formulations. The chromatographic system comprised of a LC-600 pump, SCL-6B system controller, and SPD-6AV detector (Shimadzu, Japan). The mobile phase consisted of 0.045 M ammonium sulfate in water. The chromatographic separation was achieved at ambient temperature on a Betabasic C-18 column (250×4.6 mm, Keystone Sci.). The flow rate was maintained at 0.75 ml/min and effluents are monitored at 205 nm. 4-(2-Aminoethyl)benzene sulfonamide was used as an internal standard (IS). This method required less than 7 min of chromatographic time. The standard curves were linear over the concentration range of 1–100 μg/ml for creatine and 2–100 μg/ml for creatinine, respectively. The relative standard deviations (RSD) for the within-day and day-to-day precision for creatine were within 1.0–4.6 and 2.2–4.7%, respectively. The RSD for the accuracy of creatine assay was in the range of 2.4–4.7%. The RSD values for the within-day precision, day-to-day precision and accuracy for creatinine validation were 1.7–4.4, 2.3–5.4 and 2.4–4.8%, respectively. This method was used to determine: (i) the creatine concentration in various marketed products; (ii) saturated solubility of various creatine salts; and (iii) stability of creatine in aqueous solution. In conclusion, a simple and sensitive LC method with UV detection was developed for the simultaneous determination of creatine and creatinine in formulations. Di-creatine citrate salt showed a higher aqueous solubility (at 25 °C) as compared to creatine and creatine monohydrate. Some of the over-the-counter (OTC) products tested contained a very low level of creatine in contrast to their label claim. Substantial conversion of creatine into creatinine was noticed in liquid formulation.

Liquid chromatographic determination of erythromycins in fermentation broth

A simple and sensitive isocratic LC method is described for the determination of erythromycins in fermentation broths. A simple technique utilizing acetone-methyl ethyl ketone, 1∶1, as extraction solvent was coupled with suitable chromatographic conditions—compounds were separated on a 250 mm×4.6 mm i.d., 5 μm, reversed-phase column at 65°C with acetonitrile-0.2m K2HPO4 pH7.0-water, 35:5:60 (v/v), as mobile phase at a flow rate of 1.0 mL min−1. UV detection was performed at 215 nm. Separation of erythromycin F from polar components of the fermentation liquid was sufficient. Erythromycins A, B, C, D, and E, andN-desmethylerythromycin A were also separated, as were known decomposition products of erythromycin A and several unknown components. The method is suitable for monitoring the progress of erythromycin fermentation.

Isocratic separation of erythromycin, related substances and degradation products by liquid chromatography on XTerra RP18

A simple, sensitive, selective and robust isocratic LC method is described for the analysis of erythromycin on XTerra RP18. The main component, erythromycin A, is separated from all known related substances and degradation products. Several unknown impurities are also separated. Acetonitrile-0.2 MK2HPO4pH7.0-water, (35∶5∶60, v/v) was used as a mobile phase at 1.0 mL min−1. UV detection was at 215 nm. The robustness of the method was evaluated by a full-factorial experimental design.

Fluorescence properties of carbazole- N-(2-methyl)acetyl chloride and determination of amino compounds via high-performance liquid chromatography with pre-column fluorescence derivatization

A sensitive LC method for the determination of amino compounds with fluorescence detection has been developed. The reaction of fluorescent tagging reagent, namely carbazole- N-(2-methyl)acetyl chloride (CMA-Cl), with amino acids is reported. Emission maximum for the CMA derivatives of amino acids is 360 nm ( λ ex=335 nm). In all cases, the derivatives exhibit strong fluorescence, whereas the reagent itself also exhibits fluorescence. It is found that the labelled derivatives using CMA-Cl are very stable; <4% decomposition occurs after heating at 40°C for 24 h in neutral solution. Fluorescence intensity of amino acid derivatives is higher in neutral and alkaline than in acidic solutions. This method, in conjunction with a gradient elution, offers baseline resolution of 18 amino acids including Orn from a linear acetonitrile gradient (here, Asn, Gln and Trp are not tested, the principal reasons: Gln and Asn, in a real sample hydrolysed, have been changed into Glu and Asp; Trp gives as much as 60% loss of its monosubstituted derivative under proposed derivatization conditions.). Separation of derivatives is carried out on a reversed-phase C 18 column with good reproducibility. Derivatization and chromatographic conditions are optimized. The relative standard deviations ( n=6) for 50 pmol of each amino acid derivative are <4.5%. The detection limits, calculated by the corresponding peak heights (in cm) by injecting successively lower concentrations until a signal-to-noise of 3:1, are 10–65 fmol for the labelled amino acids.

Determination of alcohols using condensation agent carbazole-9-acetyl-benzene-disulfonate by high performance liquid chromatography with pre-column fluorescence derivatization

A sensitive LC method for the determination of alcohols, using fluorescent condensation agent carbazole-9-N-acetylbenzne-disulfonate(CABS), has been developed. A mixture of alcohols and triethylamine catalyst in dichloromethane or chloroform is treated with CABS to give quantitative yields of esters. Emission maximum for the derivatized alcohols is 365 nm (λex 335 nm). The labeled derivatives are very stable, no significant decomposition is observed after heating in 40% at 40°C for 24 h. The method, in conjunction with a multigradient program, offers baseline resolution of common alcohol derivatives on a reversed-phase C18 column. Studies on derivatization conditions indicate that primary and secondary alcohols react very fast with CABS in the presence of triethylamine in dichloromethane or chloroform to give the corresponding fluorescent derivatives. This method is more convenient and more efficient than previous methods which require prior conversion of carboxylic acids to acyl chlorides. The separation of alcohol derivatives has good reproducibility and the rsd's (n=5) for 50 pmol of each alcohol are 4%. Detection limits are at the fmol level.

Characterization and application of acridine-9-N-acetyl-N-hydroxysuccinimide as a pre-column derivatization agent for fluorimetric detection of amino acids in liquid chromatography

A simple and sensitive LC method that rapidly labels amino compounds including amino acids, using acridine-9-N-acetyl-N-hydroxysuccinimide (AAHS) which was synthesized by the reaction of acridine-9-N-acetic acid with benzenedisulfonyl-N-hydroxysuccinimide, was developed. A mixture of amines is treated with AAHS in the presence of triethylamine in non-aqueous acetonitrile or in 0.2 mol l–1 borate buffer at pH 8.0–9.0 in 40% v/v acetonitrile solution to give quantitative yields of amides. The emission maximum for the derivatized amines is 435 nm (λex = 404 nm). The labeled derivatives are very stable; no significant decomposition is observed after heating in 50% acetonitrile at 40 °C for 24 h. Studies on the derivatization conditions indicate that amines or amino acids react very rapidly with AAHS under the proposed conditions. The method, in conjunction with a multi-step gradient, offers baseline resolution of common amine or amino acid derivatives on a reversed-phase C18 column. This method is more convenient and more efficient than previous methods which require prior conversion of carboxylic acids to acyl chlorides, which are unstable to moisture. The LC separation of amine or amino acid derivatives has good reproducibility. The established method is also suitable for the determination of other amine compounds in various biological fluids.

CARBAZOLE-9-N-ACETYL-N-HYDROXYSUCCINIMIDE (CAHS) AS PRE-COLUMN DERIVATIZATION AGENT FOR FLUORIMETRIC DETECTION OF AMINO COMPOUNDS WITH LIQUID CHROMATOGRAPHY

A simple and sensitive LC method for the determination of amino compounds, using carbazole-N-acetyl-N-hydroxy-succinimide (CAHS) as condensation agent, has been developed.A mixture of amines is treated with CAHS, in the presence of triethylamine catalyst in non-aqueous acetonitrile or in the presence of 0.2 M borate buffer at pH 8.0–9.0 in 40% (v/v) of acetonitrile solution, to give quantitative yields of amides. Emission maximum for the derivatized amines is 365 nm (λex 335 nm). Studies on derivatization conditions indicate that amines react very fast with CAHS under proposed conditions. The method, in conjunction with a multi-step gradient, offers baseline resolution of common amine derivatives on a reversed-phase C18 column. Derivatization reaction is more convenient and more efficient than previous methods which require prior conversion of carboxylic acids to acyl chlorides. The LC separation of amine derivatives has good reproducibility. The established method is also suitable for the determination of other amines in various biological fluids.

HPLC of amino acids and oligopeptides by pre-column fluorescence derivatization with N-hydroxysuccinimidyl-α-(9-phenanthrene)-acetate

A sensitive LC method for the detection of amino acids and oligopeptides with pre-column fluorescence derivatization has been developed. Glycine, glycylglycine, triglycine, glutathione, glutamic acid, and cysteine were separated on a reversed-phase C18 column with methanol-water-triethylamine eluent, derivatization and chromatographic conditions were optimized. The six derivatives were eluted in 20 min with good reproducibility. The relative standard derviations (n=6) at an analytical concentration of 2×10−6 M are <5%. Detection limits (signal-to-noise ratio=3) for the six derivatives are 23–68 fmol.

Multidimensional narrow bore liquid chromatography analysis of Ro 24-0238 in human plasma

A highly sensitive LC method has been developed and validated for quantitation of Ro 24-0238 in human plasma using Ro 24-2446 as an internal standard. With 1 ml of plasma, the limit of quantitation of the method was 50 pg ml −1 of Ro 24-0238. After solid-phase extraction with C 18 reversed-phase cartridges, the samples were reconstituted in an acidic buffer solution; under these conditions, Ro 24-0238 and Ro 24-2446 (IS) were converted to their cationic forms. The LC system employed a strong cation exchange column and a narrow bore reversed-phase column, connected via a column switching valve. The cationic analyte and internal standard were separated from most of the endogenous components of plasma on the cation exchange column. A small fraction containing the analyte and the internal standard was transferred by automated valve switching to the narrow bore reversed-phase column, which further resolved the individual components. The chromatography was monitored by UV absorption at 322 nm. The overall intra-assay precision was 3.6% (RSD) and the per cent error was less than ±11%. The overall inter-assay precision was 3.9% (RSD). Linearity was demonstrated in a concentration range of 50–5000 pg ml −1. This method has been applied to pharmacokinetic studies of Ro 24-0238 in man.