Liquid Chromatographic Assay Method Research Articles

Determination of ampicillin in serum by high-performance liquid chromatography with precolumn derivatization

A high-performance liquid chromatographic assay method using precolumn derivatization and fluorescence detection has been developed and validated for the determination of ampicillin in serum. The presented method is simple and provides improved selectivity and sensitivity over other existing HPLC methods. It is linear over the concentration range of 100 to 10 000 ng/ml (method 1) and 2 to 1000 ng/ml (method 2) and the extraction recovery is more than 75%. The coefficient of variation is found to be less than 10% over the concentration ranges studied.

Stereoselective high-performance liquid chromatographic assay for the determination of OPC-18790 enantiomers in human plasma and urine

A high-performance liquid chromatographic assay method for the quantification of OPC-18790 enantiomers in human plasma and urine is described. A human plasma or urine was extracted with organic solvent under alkaline conditions following the addition of internal standard. The enantiomers and internal standard were then derivatized by reaction with the chiral reagent GITC (2,3,4,6-tetra-O-acetyl-β- d-glucopyranosyl isothiocyanate), followed by octadecylsilica chromatographic separation of the diastereomeric products. The mobile phase consisted of acetonitrile—water (41:59). The fluorescence of the eluate was monitored at 355/405 nm. The lowest quantification limit of each enantiomer was 10 ng/ml in plasma and 0.1 μg/ml in urine. Both intra- and inter-day coefficients of variation were below 10%. The assay is sensitive, specific and applicable for stereoselective pharmacokinetic studies in human.

The Development of a High Performance Liquid Chromatographic Assay Method to Determine Pyruvate Generated During The C-S Lysis of Cysteine Conjugated Xenobiotics

It is now well recognized that cysteine conjugates are sometimes responsible for the mutagenic and cytotoxic consequences which occur following the ingestion of halocarbons. These toxic reactions arise from the generation of reactive metabolites via the cleavage of the C-S bond in cysteine conjugates. The enzymes which facilitate this reaction on cysteinyl conjugate metabolites are now known as C-S lyases. The products of the C-S lyase catalysis of cysteine conjugates are a reactive thiol, pyruvic acid and ammonia. Here we report the development of a sensitive fluorometric hplc assay for pyruvic acid using the derivatising agent 1,2-phenylenediamine. It was found that using this assay CSL activity could be detected in a porcine C-S lyase preparation. It was also observed that this assay was approximately ten-fold more sensitive than the existing spectrophotometric assay. It is now well recognized that cysteine conjugates are sometimes responsible for the mutagenic and cytotoxic consequences which occur following the ingestion of halocarbons. These toxic reactions arise from the generation of reactive metabolites via the cleavage of the C-S bond in cysteine conjugates. The enzymes which facilitate this reaction on cysteinyl conjugate metabolites are now known as C-S lyases. The products of the C-S lyase catalysis of cysteine conjugates are a reactive thiol, pyruvic acid and ammonia. Here we report the development of a sensitive fluorimetric hplc assay for pyruvic acid using the derivatizing agent 1,2-phenylenediamine. It was found that using this assay CSL activity could be detected in a porcine C-S lyase preparation. It was also observed that this assay was approximately ten-fold more sensitive than the existing spectrophotometric assay.

Modification of a high-performance liquid chromatographic method for assay of homocysteine in human plasma

A modification of a previously published method for analysis of total homocysteine in human serum is presented. The modification was implemented to allow use of a different derivatizing agent ( i.e., 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonamide) which reacts much faster than the original derivatizing reagent and at a lower temperature. Shorter reaction time and lower temperature lead to less destruction of some biological thiols. In order to retain an isocratic mobile phase with the new derivatizing agent, a different concentration of acetonitrile was found that affords a 7–8 min retention time.

Improved high-performance liquid chromatographic determination of biotinidase activity with biotinyl-6-aminoquinoline as substrate

An improved high-performance liquid chromatographic assay method for biotinidase activity was developed using the fluorimetric substrate biotinyl-6-aminoquinoline, which was found to be more specific than the biotinyl-4-aminobenzoate previously used. The new method measures the intensity of the fluorescent signal at wavelengths (excitation 350 nm; emission 550 nm) longer than those (excitation 276 nm; emission 340 nm) for 4-aminobenzoate. The analysis of fluorescence in the visible spectrum reduced considerably the number of interfering peaks compared with analysis in the ultraviolet region. This method also made it possible to measure the biotinidase activity directly in samples usually difficult to calculate, such as human and bovine milk or porcine serum; the use of biotinyl-6-aminoquinoline allowed the analysis of the enzyme reaction in milk and porcine serum without pretreatment or dialysis. Stoichiometric increase and decrease of the substrate and product, respectively, were demonstrated. Michaelis constants for biotinyl-6-aminoquinoline were measured at various stages of partial purification. Because the solubility of these synthetic substrates in the aqueous phosphate buffer is limited, the determination of both Michaelis constant and maximum velocity by extrapolation may be helpful for the characterization of the kinetics of biotinidase.

High-Performance Liquid Chromatographic Method for Assay of Otilonium Bromide, Diazepam, and Related Compounds in Finished Pharmaceutical Forms

A rapid, simple, stability-indicating assay procedure for otilonium bromide, a smooth muscle relaxant agent, and diazepam in composite tablet analysis was developed with high-performance liquid chromatography. The tablet matrix was dissolved with water, and drugs were extracted with acetonitrile containing an internal standard. An aliquot was centrifuged and chromatographed on a 5μm, reversed- phase column with 0.5M sodium acetate trihydrate buffer containing 5mM 1-heptanesulfonic acid monohydrate sodium salt:methanol (30:70; v/v; adjusted to pH 6.0 with glacial acetic acid) as the mobile phase. The selectivity of the chromatographic system was demonstrated by resolving both compounds from various potential degradation products of each compound. The method is linear, quantitative, and reproducible.

Improved high-performance liquid chromatographic assay method for the enantiomers of ibuprofen

A rapid, inexpensive and sensitive high-performance liquid chromatographic method for the quantitation of ibuprofen enantiomers from a variety of biological fluids is reported. This method uses a commercially available internal standard and has significantly less interference from endogenous co-extracted solutes than do previously reported methods. The method involves the acid extraction of drug and internal standard [(±)-fenoprofen] from the biological fluid with isooctane—isopropanol (95:5) followed by evaporation and derivatization with enthylchloroformate and R-(+)-α-phenylethylamine. Excellent linearity was observed between the peak-area ratio and enantiomer concentration ( r > 0.99) over a concentration range of 0.25–50 μg/ml. This method is suitable for the quantitation of ibuprofen from single-dose pharmacokinetic studies involving either rats or humans.

Liquid chromatographic separation of the anomers and isomers of 2′-deoxyuridine

A liquid chromatographic assay method was developed to separate 2′-deoxyuridine from its anomer and pentopyranosyl isomers. The influence of the pH of the mobile phase, the type of organic modifier and the column temperature on the chromatographic parameters was systematically investigated. Using a Hypersil C18 (5 μm) column at 10°C, the following mobile phase was found suitable: tetrahydrofuran −0.2 M potassium phosphate buffer pH 4.0-water (0.1∶5∶94.9, v/v).

Determination of trichlormethiazide in human plasma and urine by high-performance liquid chromatography

This paper describes a high-performance liquid chromatographic (HPLC) assay method for the determination of trichlormethiazide (TCM) in human plasma and urine. After extraction and separation on an ODS column TCM from plasma was detected by oxidation in an electrochemical detector (ECD) by a porous graphite electrode. The sensitivity was better than HPLC with UV detection, enabling the determination of 2 ng ml−1 TCM in human plasma. This method also allows determination of TCM at higher concentrations by exchanging the UV for the electrochemical detector. To study the pharmacokinetics, TCM in plasma and urine was assayed with coefficients of variation in the range 2–3%. The method has the advantages of high sensitivity for plasma assay and high precision with a simple procedure for both plasma and urine samples. Small samples of 0.5 ml plasma per assay also reduced the total volume of plasma needed.

Liquid chromatography of erythromycin A and related substances on poly(styrene-divinylbenzene)

An isocratic liquid chromatography method for assay and purity control of erythromycin is presented. Erythromycin A is separated from all its potential impurities, except erythromycin D. The selectivity depends on the pore size of the poly(styrene-divinylbenzene) stationary phase. Wide pore PLRP-S 8 μm 1000 A shows the best selectivity. The column is heated at 70°C. The mobile phase is acetonitrile-2-methyl-2-propanol-0.2 M potassium phosphate buffer pH 9.0-water (3:16.5:5:75.5). The flow rate is 2 ml/min. UV detection is performed at 215 nm. The total analysis time is about 30 min. The method was used to compare official standards.

Liquid Chromatography of Pesticide Formulations Containing Dicamba, 2,4-D, and MCPP

Abstract The present paper describes a reverse phase liquid chromatographic method for assay of samples that combine dicamba, 2,4-D, and MCPP In herbicide formulations. Samples are diluted with isopropanol-water (2 + 1) and chromatographed on a C-18 column with a gradient mobile phase of increasing acetonitrlle concentration with 2% acetic acid solution as the aqueous phase. After an initial hold time of 5 min, the gradient is increased from 25 to 40% acetonitrlle over 10 min. With a flow rate of 1.5 mL/mln, dicamba, 2,4-D, and MCPP elute at 6, 13, and 16.5 min, respectively. The wlthin-day coefficient of variation was 0.1 to 3.2%; between-day variation was 0.6 to 5.5%. The method separates the 3 herbicides from major potential impurities. This method differs from the AOAC official method in that it uses 2% acetic acid in the mobile phase, a gradient flow, no Internal standard, and a data handling system to quantitate the 3 herbicides from the same injection.

High-performance liquid chromatographic method for the simultaneous assay of a new synthetic penem molecule and its salt-forming agent in injectable formulations

A rapid, stability-indicating, reversed-phase high-performance liquid chromatographic method for the direct and simultaneous determination of a new β-lactam molecule and its salt-forming agent in finished dosage forms was studied for the development of injectable formulations.

CHEMICAL STABILITY OF CEFOTETAN DISODIUM IN 5% DEXTROSE AND 0.9% SODIUM CHLORIDE INJECTIONS

The chemical stability of cefotetan disodium in 5% dextrose and 0.9% sodium chloride injections has been studied using a stability-indicating high-pressure liquid chromatographic (HPLC) assay method. The drug appears to be relatively unstable at 25 degrees C (expiry time 2 days), compared with at least 41 days at 5 degrees C and at least 60 days at -10 degrees C. Thawing the frozen samples in a microwave (90 s) did not cause any significant decomposition. The manufacturer's recommended expiry time of 4 days at 5 degrees C and at least 7 days at -10 degrees C is very conservative. The HPLC method developed is accurate and precise with a relative percentage standard deviation of 1.7 based on six readings. The method appears to be stability-indicating as the samples decomposed under drastic conditions had almost no drug left and new peaks were observed in the chromatograms.

Validation of an amperometric high-performance liquid chromatographic determination of epinephrine in bupivacaine and epinephrine injection

Epinephrine, as the bitartrate salt at a 1:200000 dilution is contained in the 0.5% bupivacaine HCl, 2-piperidinecarboxamide, 1-butyl-N-(2,6-dimethylphenyl)-, monohydrochloride monohydrate. The development and validation of a sensitive and specific stability-indicating high-performance liquid chromatography (HPLC) assay method utilizing amperometric detection was desired as an improvement over and simplification of previous methods for epinephrine in this product

High-performance liquid chromatographic analysis of imidazolium and pyridinium oximes in plasma and urine

Imidazolium oximes are useful in the treatment of organophosphorus agent poisoning. However, the extant methods for analyzing oximes in plasma and urine samples are not adequate. A unique high-performance liquid chromatographic assay method was developed for quantitating either imidazolium or pyridinium oximes. Plasma or urine samples can be injected directly onto the column after a centrifugation filtration step. This method demonstrates a different approach in the method development for quaternary ammonium compounds using non-end-capped reversed-phase columns and a mobile phase containing competing cations. In addition, a preliminary pharmacokinetic study of the imidazolium oxime in rabbits was carried out using this method.

Knowledge-based expert system for troubleshooting high-performance liquid chromatographic assay methods

An expert system has been written to assist in troubleshooting 21 high-performance liquid chromatographic (HPLC) assay methods frequently performed in our laboratories. The knowledge used in this expert system includes the facts, heuristics (rules-of-thumb based on experience that provides short cuts over a strict textbook approach to a problem) and problem-solving strategies for diagnosing problems that occur in HPLC assay methods. The expert system operates by gathering necessary data from a user, analyzing the assay parameter, determining the most probable causes of the problem and displaying its conclusions together with justifications for its diagnosis. Data are quickly and easily entered by a user through the use of a notebook-style form that is displayed on a PC screen. The expert system analyzes the assay problem based on user-supplied chromatographic symptoms and selectivity, capacity factor ( k′) and resolution (itR s that are derived from the data. Once the system has diagnosed the most probable causes, it prints these out ranked in order of most likely causes and proceeds for further in-depth troubleshooting.

High-performance liquid chromatography method for assay of diltiazem hydrochloride and its related compounds in bulk drug and finished tablets.

The method provides for the resolution of trans-diltiazem and seven known and several unknown related compounds from diltiazem HCl. Minimum detectable amounts were less than 0.1%, except for an intermediate which originates early in the synthetic process, for which the sensitivity is approximately 2%. The relative standard deviation of the assay procedure is 0.15%. Total related compounds in four bulk drug and four tablet samples were less than 0.25%. The specific rotation of four samples of diltiazem HCl analyzed in duplicate was between +112 and +114 degrees. The UV absorption spectra of all compounds exhibited two maxima, one between 203 and 213 nm and the other between 230 and 244 nm.

Quick and Simple Determination of Dihydroergotamine by High-Performance Liquid Chromatography

Abstract A simple and rapid liquid chromatographic assay method using a fluorescence detector for quantitation of dihydroergotamine in plasma without extraction was developed. After precipitating the protein with acetonitrile, the supernatant liquid was directly injected for analysis. Chromatographic separation was achieved on C18 reversed phase column and the mobile phase was the isocratic mixture of methanol, acetonitrile and glycine buffer (0.5:3.5:6.0). With this eluting solvent the drug and its internal standard were well separated from the interference of the plasma sample. The average recovery of dihydroergotamine from 6 replicate samples of different concentrations (5-30 ng/ml) were 92.2 ± 3.37%. The minimum amount of dihydroergotamine detectable by this method was 2 ng/ml of sample.

The simultaneous assay of riboflavin 5-phosphate sodium and other water-soluble vitamins in liquid multivitamin formulations by liquid chromatograph

A simple liquid chromatographic method for the simultaneous assay of niacinamide, pyridoxine, thiamine and riboflavin 5'-phosphate sodium in liquid multivitamin formulations is described. A novel aspect of the procedure is the conversion of the riboflavin 5'-phosphate sodium to riboflavin (vitamin B(2)) by reaction with alkaline phosphatase prior to quantitative HPLC. Excellent recovery, linearity and accuracy are obtained by the proposed method.

Novel dual-wavelength monitoring approach for the improved rapid separation and estimation of adenine nucleotides and creatine phosphate bu high-performance liquid chromatography

A rapid and improved method for the simultaneous quantification of adenine nucleotides, including adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, as well as creatine phosphate by high-performance liquid chromatography is described. A programmable multi-wavelength UV detector was used to monitor the effluent initially at 210 nm for 4 min to measure creative phosphate, and then at 259 nm for 6 min to quantitate adenosine mono-, di- and triphosphate. The method is, thus, not only rapid, but also extremely sensitive because of the utilization of corresponding absorption maxima for creatine phosphate and adenine nucleotides, rather than measuring all of them at a single wavelength. In addition, computer-generated standard curves were used to estimate these compounds, thereby improving the accuracy of the measurements. Since myocardial high-energy phosphate levels are widely measured because they are reliable parameters for myocardial preservation during ischemia, these compounds were assayed in pig heart prior to and following ischemia. The results were compared with those obtained by conventional high-performance liquid chromatographic assay methods in order to examine the validity of this method. Our results indicate that this is indeed a novel method, which is more rapid as well as sensitive and accurate compared with other methods.