High-performance Liquid Chromatographic Assay Method Research Articles

HPLC를 이용한 어류 중의 Oxolinic Acid 분석

A high-performance liquid chromatography assay method for oxolinic acid in fish products was developed, evaluated and validated through the monitoring of oxolinic acid based on farming and distribution. The recovery rate of the developed method was <TEX>$102.3-106.7\%$</TEX> as compared to conventional methods. The stock solution was stable for 3 weeks under refrigerated condition at <TEX>$4^{\circ}C$</TEX> The performance limit was evaluated as 0.01ppm of oxolinic acid in fish muscle. 478 fish samples such as olive flounder, genuine porgy, common sea bass and black rock fish collected from fish farms in the coastal area from September 2001 to October 2004 were analyzed to evaluate overall efficiency of the modified method and to monitor the actual condition of oxolinic acid usage in fish farm. According to the monitoring results, the modified method was suitable for analysis of oxolinic acid in fish muscle and oxolinic acid might be used in a small portion of fish farms. The suggested analysis method of oxolinic acid was registered in the Korean Official Methods of Food Analysis and is being utilized for fishery products by the Korea Food and Drug Adminstration and the National Fisheries Products Quality Inspection Service.

A fluorescence-based assay for indoleamine 2,3-dioxygenase

A rapid and sensitive fluorescence-based bioassay for determination of indoleamine 2,3-dioxygenase (IDO) activity has been developed. This assay relies on the quantification of the amount of kynurenine produced in the assay medium by fluorescence and complements the standard absorbance and high-performance liquid chromatography (HPLC) assay methods. The fluorescence method has limits of detection similar to those of the standard assay methods. Measured activities of IDO, including in the presence of tryptophan-based inhibitors, were in statistical agreement with the absorbance and HPLC assay methods. The fluorescence-based assay was also suitable for assessment of IDO inhibition by compounds that are incompatible with the absorbance method.

ICH guidance in practice: establishment of inherent stability of secnidazole and development of a validated stability-indicating high-performance liquid chromatographic assay method

The degradation behaviour of secnidazole was investigated under different stress degradation (hydrolytic, oxidative, photolytic and thermal) conditions recommended by International Conference on Harmonisation (ICH) using HPLC and LC–MS. A stability-indicating HPLC method was developed that could separate drug from degradation products formed under various conditions. Secnidazole was found to degrade significantly in alkaline conditions, oxidative stress, and also in the presence of light. Mild degradation of the drug occurred in acidic and neutral conditions. The drug was stable to dry heat. Resolution of drug and the degradation products formed under different stress studies were successfully achieved on a C-18 column utilizing water–methanol in the ratio of 85:15 and at the detection wavelength of 310 nm. The method was validated with respect to linearity, precision (including intermediate precision), accuracy, selectivity and specificity.

Validated specific HPLC methods for determination of prazosin, terazosin and doxazosin in the presence of degradation products formed under ICH-recommended stress conditions

The present paper describes development of stability-indicating high-performance liquid chromatographic (HPLC) assay methods for three alpha-adrenergic-blocker drug substances, namely, prazosin, terazosin and doxazosin, in the presence of degradation products generated from forced decomposition studies. Resolution of drugs from degradation products was obtained using a reversed-phase C-18 column using water/acetonitrile/methanol/glacial acetic acid/diethylamine (25:35:40:1:0.017) as mobile phase for prazosin and terazosin and acetonitrile/water/glacial acetic acid/diethylamine (65:35:1:0.02) for doxazosin. The detection was done at 254 nm. The methods were validated with respect to linearity, precision, accuracy, specificity and robustness.

Quantitation of tolmetin by high-performance liquid chromatography and method validation.

A high-performance liquid chromatographic (HPLC) assay method for assessing the degradation of tolmetin (TLM) is developed and validated under acidic, basic, and photoirradiated conditions. The HPLC method includes an Inertsil 5 ODS-3V column (250- x 4.6-mm i.d.), guard column of Inertsil 7 ODS-3V (50- x 4.6-mm i.d.), mobile phase of CH(3)OH-1% HOAc (64:36, v/v), and UV detection at 254 nm. The developed method satisfies the system suitability criteria, peak integrity, and resolution for the parent drug and its degradants. The established assay method exhibits good selectivity and specificity suitable for stability measurements. From the intra- and interday tests of six replicates, the coefficients of variation are between 0.20% and 1.77% for the former, and 0.12% and 3.40% for the latter. Recoveries are found to be 98.7-101.7%. TLM is determined to be more reactive when exposed to light and acidic conditions, yet TLM is stable in a basic medium. A kinetic study of the photodegradation of TLM shows that it follows an apparent first-order reaction in three alcoholic solvents.

A fluorescence-based, high-performance liquid chromatographic assay to determine acid sphingomyelinase activity and diagnose types A and B Niemann–Pick disease

Acid sphingomyelinase (ASM; sphingomyelin phosphodiesterase, EC 3.1.4.12) is the lysosomal enzyme that hydrolyzes sphingomyelin (SPM) to phosphorylcholine and ceramide. An inherited deficiency of ASM activity results in Types A and B Niemann–Pick disease (NPD). In this study we report a new assay method to detect ASM activity and diagnose NPD using the fluorescent substrate BODIPY C12-SPM and reverse-phase high-performance liquid chromatography (HPLC). The reaction product, BODIPY C12-ceramide (B12Cer), could be clearly and efficiently separated from the substrate within 4 min using a reverse-phase column (Aquasil C18, Keystone Scientific). Femtomole quantities of B12Cer could be detected in as little as 1.0 μl of human plasma, providing a sensitive measure of ASM activity. The mean ASM activity in human plasma from NPD patients (36 pmol/ml/h) was only 2.7% of that in normal plasma (1334 pmol/ml/h), confirming the specificity and diagnostic value of this new assay method. Importantly, the mean ASM activity in human plasma from NPD carriers (258.3 pmol/ml/h) also was significantly reduced (19.5% of normal). The ranges of ASM plasma activities in NPD patients ( N=19), NPD carriers ( N=11), and normal subjects ( N=15) were 2.5–97.3, 108–551, and 1030–2124 pmol/ml/h, respectively. Based on these results, we suggest that this fluorescence-based HPLC assay method is a reliable, rapid, and highly sensitive technique to determine ASM activity and that plasma is a very reliable and simple source for the accurate diagnosis of NPD patients and carriers based on ASM activity.

High-performance liquid chromatographic assay with fluorescence detection for the simultaneous measurement of carboxylate and lactone forms of irinotecan and three metabolites in human plasma

Irinotecan (CPT-11), a camptothecin analog, is metabolized to SN-38, an active topoisomerase I inhibitor, and inactive metabolites, including APC and SN-38 glucuronide (SN-38G). A high-performance liquid chromatographic assay method to simultaneously measure the lactone and carboxylate forms of CPT-11, SN-38, SN-38G, and APC in human plasma was developed. Chromatography was accomplished with a reversed-phase C 8 column and fluorescence detection. A gradient mobile phase system was used. The buffer for mobile phase A consisted of 0.75 M ammonium acetate, 5 m M tetrabutylammonium phosphate (pH 6.0), and acetonitrile (86:14, v/v). The buffer for mobile phase B was identical to mobile phase A with the exception of the concentration (50:50, v/v). Precipitation of plasma proteins was performed with cold methanol. The linear range of detection of the lactone and carboxylate forms of SN-38, SN-38G, and APC was 2–25 ng/ml, and 5–300 ng/ml for CPT-11. The limit of quantitation for the analytes ranged from 0.5 to 5 ng/ml. Analysis of patients’ plasma samples obtained before and after CPT-11 administration showed that the assay is suitable for measuring lactone and carboxylate forms of CPT-11, SN-38, SN-38G, and APC in clinical studies.

Kinetics of kavain and its metabolites after oral application

Kavain metabolism in humans was the target of this current investigation. In the present study a high-performance liquid chromatographic (HPLC–DAD) assay method for the simultaneous determination of kavain and its main metabolites ( p-hydroxykavain, p-hydroxy-5,6-dehydrokavain and p-hydroxy-7,8-dihydrokavain) in serum and urine was developed and validated. The metabolites were mainly excreted in the form of their conjugates. All kavain metabolites were detectable in serum and urine, except for p-hydroxy-7,8-dihydrokavain, which was found in urine only. Confirmation of the results and identification of the metabolites were performed by LC–MS or LC–MS–MS. Kinetics of kavain and its metabolites in serum were investigated after administration of a single oral dose (800 mg kavain). Within 1 and 4 h after uptake, the serum concentrations ranged between 40 and 10 ng/ml for kavain, 300 and 125 ng/ml for p-hydroxykavain, 90 and 40 ng/ml for o-desmethyl-hydroxy-5,6-dehydrokavain, and 50 and 30 ng/ml for 5,6-dehydrokavain.

LC fluorescence method for multiple synthetic compounds to rapidly create in vivo pharmacokinetic database utilizing 'N-in-One' dosing.

This manuscript reports development and validation of an assay method in rat serum for the simultaneous estimation of C1, C2, and C3, in-house CDRI molecules, of the class of aryloxy-substituted aryl-piperazinyl derivatives. The assay was applied to determine pharmacokinetic data after simultaneous intravenous administration of these three compounds. A high-performance liquid chromatography assay method using isocratic elution and fluorescence (excitation, 250 nm; emission 350 nm) was developed for simultaneous estimation of all the three compounds in rat serum. Linearity was observed between 12.5 and 400 ng/ml for all the three compounds in serum. Recoveries were highly consistent over the concentration ranges for all the analytes. Variations in the intra- and inter-batch accuracy and precision were within the acceptable limits of +/-20% at the limit of quantitation, whereas at higher concentrations it was +/-15%. A mixture of the three compounds was administered intravenously to rats. Blood samples were collected over a period of 6 h and analyzed to determine serum levels and pharmacokinetics of each compound. The pharmacokinetics of the aforementioned three compounds was also determined after individual administration. The results obtained in the N-in-One dosing correlated well with discrete dosing of compounds. Based on the results obtained, C2 emerges to be the compound with appropriate pharmacokinetic parameters. Thus, the N-in-One method is a useful method for increasing the throughput to obtain the pharmacokinetic information.

High-performance liquid chromatographic assay with fluorescence detection for the determination of cephaeline and emetine in human plasma and urine

A high-performance liquid chromatographic assay method for the quantitation of ipecac alkaloids (cephaeline and emetine) in human plasma and urine is described. Human plasma or urine was extracted with diethylether under alkaline conditions following the addition of an internal standard. Concentrations of alkaloids and internal standard were determined by octadecylsilica chromatographic separation (Symmetry C 18 columns, plasma analysis; 15 cm×4.6 mm I.D., 5 μm particle size, urine analysis; 7.5 cm×4.6 mm I.D., 5 μm particle size). The mobile phase consisted of buffer (20 mmol/l 1-heptanesulfonic acid sodium salt, adjusted to pH 4.0 with acetic acid)–methanol (51:49, v/v). Eluate fluorescence was monitored at 285/316 nm. The lowest quantitation limits of cephaeline and emetine were 1 and 2.5 ng/ml, respectively, in plasma, and 5 ng/ml in urine. Intra- and inter-day relative standard deviations were below 15%. The assay is sensitive, specific and applicable to pharmacokinetic studies in humans.

Anion-exchange high-performance liquid chromatography assays of plasma lipoproteins and modified low-density lipoproteins using a ProtEx-DEAE column

Previously [Anal. Biochem., 232 (1995) 163–171], we reported a high-performance liquid chromatography (HPLC) assay method for human plasma lipoproteins using a diethylaminoethyl (DEAE)-glucomannan column, which is not commercially available. In this study, HPLC assay methods for lipoproteins in plasma samples of human and experimental animals, and modified low-density lipoproteins (LDLs) of rabbits have been developed using a commercially available anion-exchange ProtEx-DEAE column. For the assays of plasma lipoproteins, the method includes complete separation of high-density lipoproteins, LDLs and very low-density lipoproteins within 20 min using stepwise elution, and determination by post-column reaction with an enzymatic cholesterol reagent as the total cholesterol (TC) level. Similarly, mild oxidative and artificially oxidised LDLs were separated into their subfractions using stepwise elution, and determined based on the TC level. The methods using the DEAE-glucomannan and ProtEx-DEAE columns were cross-validated. There was an excellent correlation between the two methods. The obtained results reveal that the anion-exchange HPLC method using the ProtEx-DEAE column could be useful for the assays of plasma lipoproteins and modified LDLs.

Stability-indicating high-performance liquid chromatographic assay method and photostability of carprofen.

A rapid, sensitive, and accurate stability-indicating high-performance liquid chromatographic assay method for determining the degradation of carprofen (CPF) is developed and validated under acidic, basic, or photo-irradiated conditions. The analysis is monitored with a Cosmosil 5C18-AR column using a mobile phase of CH3CN-H2O-AcOH (50:49:1, v/v/v) at 260 nm. The developed method satisfies the system suitability criteria, peak integrity, and resolution among the parent drug and its degradation products. The results indicate that the established assay method shows good selectivity and specificity suitable for stability measurements of CPF. CPF is found to be more sensitive to exposure to light and in acidic conditions, but it is stable in a basic medium. The kinetic study of the photodegradation of CPF follows an apparent first-order reaction in a variety of solvents. The solvent effects on the rates of degradation are in the decreasing order of chloroform > dichloromethane > methanol > ethanol > 2-propanol, which is irrelevant to the dielectric constant epsilon. However, the hydrogen-donating ability of the solvents is essential to the photochemical decomposition of CPF. A plot of log k versus the Kirkwood function exhibits a linear relationship in aqueous ethanolic solutions, which implies that degradation proceeds via an ionic mechanism.

Fast isocratic high-performance liquid chromatographic assay method for the simultaneous determination of gabapentin and vigabatrin in human serum

Fast isocratic high-performance liquid chromatographic assay method for the simultaneous determination of gabapentin and vigabatrin in human serum

Validation of high-performance liquid chromatographic assay methods for the analysis of carboplatin in plasma ultrafiltrate

Validation of two HPLC assays for the quantitation of carboplatin in human plasma ultrafiltrate is described. Both assay methods employed a YMC ODS-AQ 3.9×150 mm (3 μm) column for the chromatographic separation. The first method utilized direct UV detection, the second method utilized UV detection following post-column derivatization with sodium bisulfite. Structural analogues of carboplatin were synthesized and used as internal standards for the assays. With direct UV detection, sample clean-up using solid-phase extraction on amino cartridges was required prior to injection, with extraction recoveries ranging from 80 to 90%. This extraction procedure was not necessary with the post-column reaction method, which employed a more selective analytical wavelength. Unfortunately, instability of the post-column reagent was a problem and led to greater variability in predicted concentration values. For standard curves, a weighted (1/ y 2) regression approach was used for plots of peak area or peak height ratio (carboplatin/internal standard) vs. carboplatin concentration. The limit of detection of both assays was 0.025 μg/ml and both were validated for carboplatin concentrations from 0.05 to 40 μg/ml. Accuracy and precision data were generated using three batches of validation samples, each batch consisting of a standard curve and five sets of quality control samples. Stability of carboplatin in blood, plasma, plasma ultrafiltrate, and reconstituted extracts was evaluated. The assay methods were employed for the pharmacokinetic analysis of blood samples drawn from a pediatric patient that received a 400 mg/m 2 dose of carboplatin.

Therapeutic drug monitoring of tacrine: simple and fast high-performance liquid chromatography assay method for its determination in human plasma.

A new high-performance liquid chromatography (HPLC) assay method was developed for the therapeutic monitoring of tacrine. The method involved a simple protein precipitation by means of either acetonitrile or cold methanol followed by a fast isocratic separation on a CN column eluted in reversed-phase mode. The entire sample preparation took place in an HPLC vial and no further liquid transfer was required. The validation data showed that the assay method was precise, accurate, and robust. Analysis of more than 1,000 plasma samples collected from patients with Alzheimer disease demonstrates the suitability of the assay.

Stability-indicating high-performance liquid chromatographic assay of buspirone HCl.

The United States Pharmacopoeia high-performance liquid chromatographic (HPLC) assay method of buspirone is not able to discriminate buspirone from its degradation products. The purpose of this work is to develop a sensitive, selective, and validated stability-indicating HPLC assay for the analysis of a buspirone hydrochloride in a bulk drug. Buspirone HCI and its potential impurities and degradation products are analyzed on an Ultrasphere C18 column heated to 40 degrees C using a gradient program that contains monobasic potassium phosphate buffer solution (pH 6.9) and acetonitrile-methanol mixture (13:17) of 35% for 5 minutes, then increased to 54% in 5.5 minutes. The samples are monitored using a photo-diode array detector and integrated at 244 and 210 nm. The stress testing of buspirone HCI shows that buspirone acid hydrochloride is the major degradation product. The developed method shows a separation of buspirone degradation product and its potential impurities in one run. The stability of buspirone HCI is studied under accelerated conditions in order to provide a rapid indication of differences that might result from a change in the manufacturing process or source of the sample. The forced degradation conditions include the effect of heat, moisture, light, acid-base hydrolysis, sonication, and oxidation. The compatibility of buspirone HCI with some pharmaceutical excipients is studied under stress conditions. The linear range of buspirone HCI is between 5 and 200 ng/microL with a limit of quantitation of 2.5 ng/microL. The intraassay percentage deviation is not more than 0.38%, and the day-to-day variation was not more than 0.80%. The selectivity, repeatability, linearity, range, accuracy, sample solution stability, ruggedness, and robustness show acceptable values.

Simultaneous quantitation of etoposide and its catechol metabolite in human plasma using high-performance liquid chromatography with electrochemical detection

Etoposide, a highly active and widely used antineoplastic agent, is O-demethylated to its active catechol metabolite. A high-performance liquid chromatographic assay method for the simultaneous quantitation of etoposide and etoposide catechol in human plasma was established. Etoposide and etoposide catechol were extracted from plasma using chloroform and methanol followed by phase separation, evaporation of the organic phase, and reconstitution of the residue. Chromatography was accomplished using a reversed-phase phenyl analytical column (390 mm×3.9 mm I.D.) with a mobile phase of 76.6% 25 m M citric acid–50 m M sodium phosphate (pH 2.4)–23.4% acetonitrile pumped isocratically at 1 ml/min with electrochemical detection. The limit of detection for etoposide was 1.2 n M and for etoposide catechol was 0.2 n M. The precision (CV) for etoposide ranged from 0.7 to 3% and for the catechol metabolite from 1 to 6%; accuracy of predicted values ranged from 97 to 106% and 94 to 103%, respectively. The assay was linear from 0.1 to 10 μ M for etoposide and from 0.005 to 0.5 μ M for etoposide catechol in plasma. Recovery of etoposide and etoposide catechol ranged from 93 to 95% and 90 to 98%, respectively. Stability of etoposide and etoposide catechol in human plasma containing ascorbic acid stored at −70°C for one year was demonstrated. This assay procedure is suitable for evaluation of etoposide and etoposide catechol pharmacokinetics in plasma following etoposide administration.

Aminoglycoside antibiotics--two decades of their HPLC bioanalysis.

Several reviews have been published on high-performance liquid chromatographic (HPLC) methods for the determination of aminoglycoside antibiotics (aminoglycosides) in biological fluids [e.g. Nilsson-Ehle, I. (1983). J. Liq. Chromat. 6: 251]. Of these, the paper by Maitra et al. [(1979a). Clin. Chem. 25: 1361.] briefly summarizes the early 2-3 years of experience on HPLC assaying of amikacin, gentamicin, netilmicin and tobramycin in body fluids. The reviews by Nilsson-Ehle, I. [(1983). J. Liq. Chromat. 6: 251] and by Miner, D. J. [(1985). Antibiotics. In Therapeutic Drug Monitoring and Toxicology by Liquid Chromatography, (Wong S. H. Y., ed.), ch. 10, p. 269. Marcel Dekker, New York and Basel.] devoted to the monitoring of antibiotics, also evaluated the first 6-8 years of the application of HPLC assays for the aminoglycosides amikacin, gentamicin, netilmicin, sisomicin and tobramycin. This report presents a great majority of the HPLC assay methods published during the last two decades for determining practically a dozen different aminoglycoside antibiotics in body fluids, particularly in the serum or plasma, and in urine.

Effect of Various Salts on the Stability of Lansoprazole, Omeprazole, and Pantoprazole as Determined by High-Performance Liquid Chromatography

A fast and reproducible reverse-phase high-performance liquid chromatography (HPLC) assay method has been developed for the simultaneous quantitation of omeprazole, lansoprazole, and pantoprazole. The three compounds were monitored at 280 nm using Zorbax Eclipse XDB C8 (5 μm, 150 cm × 4.6 mm i.d.) and a mobile phase consisting of 700:300 phosphate buffer:acetonitrile with the pH adjusted to 7.0 with phosphoric acid. The method was used to study the effect of pH and various salts on the stability of the three compounds. The pH rate profile curve showed that pantoprazole was the most stable compound and lansoprazole the least stable. The stabilities of the compounds in salt solutions were found to be in the following order: phosphate buffer < trisodium citrate < citrate buffer ≤ acetate buffer < citric acid ≤ monosodium citrate ≤ calcium carbonate < sodium bicarbonate < sodium chloride < water. The rate of degradation had a direct relationship with the H+ and salt concentration.

Anion-exchange high-performance liquid chromatographic assay of plasma lipoproteins of rabbits, rats and mice

A fast, accurate and precise high-performance liquid chromatographic assay method has been developed for plasma lipoproteins of experimental animals, rabbits, rats and mice. The method includes complete separation of high, low and very low density lipoproteins from one another within 20 min by a DEAE–glucomannan gel using stepwise elution, and determination by postcolumn reaction with an enzymatic cholesterol reagent as the total cholesterol level. The relative standard deviation of each lipoprotein assay was highly reproducible, being less than 2.0 and 2.4% for repeatability and intermediate precision, respectively. The method was successfully applied to the assays of plasma lipoproteins in three species of normolipidemic and hyperlipidemic animals.