Simultaneous determination of diastereomers (+)-licarin A and isolicarin A from Myristica fragrans in rat plasma by HPLC and its application to their pharmacokinetics

A rapid and simple high-performance liquid chromatographic (HPLC) method was developed and validated to simultaneously analyze the diastereomers of (+)-licarin A and isolicarin A in rat plasma after intravenous administration. The analytes were extracted from the plasma by solid-phase extraction (SPE). Diastereomeric separation and determination were successfully achieved using a Diamonsil ODS C (18) reversed-phase column (250 mm x 4.6 mm i. d., 5 microm) with an RP18 guard column (8 mm x 4.6 mm i. d., 5 microm) and a mobile phase of MeOH-H (2)O (4 : 1, v/v). UV detection was at 270 nm. The linear ranges of the standard curves were 0.25 - 150.00 microg/mL for (+)-licarin A and 0.10 - 75.00 microg/mL for isolicarin A. The lower limits of detection and quantification were 0.05 and 0.25 microg/mL for (+)-licarin A, and 0.05 and 0.10 microg/mL for isolicarin A, respectively. This assay method was successfully applied to study the pharmacokinetics of diastereomers (+)-licarin A and isolicarin A in rat plasma.     

HPLC determination of dexamethasone in human plasma and its application to an in vitro release study from endovascular stents

A simple, accurate and precise HPLC assay was developed and validated for determination of dexamethasone in human plasma. Triamcinolone acetonide was used as internal standard (I.S.) and plasma samples were extracted using liquid-liquid extraction with ethyl acetate. Chromatography was performed on a C-18 column with acetonitrile-triple distilled water (28:72% v/v, pH adjusted to 2.3 with phosphoric acid) at a flow rate of 1.2 mL/min and detection wavelength 254 nm. The assay was linear at a concentration range of 0.25-6 microg/mL with recoveries >77%. Precision and accuracy were within the acceptable limits. The method was used to determine dexamethasone release from different material coated endoluminal vascular stents in in vitro human plasma experiments. The results were useful in identifying a good coating material for the stents.     

Heterogeneous effects of ethylenebisdithiocarbamate (EBDC) pesticides on oxidative metabolism of xenobiotics

Comparative evaluation of the acute effects of ethylenebisdithiocarbamate (EBDC) fungicides, mancozeb and zineb on microsomal mixed function oxidases (MFO) revealed marked substrate-dependent inhibition of oxidative metabolism of aminopyrine, p-nitroanisole and aniline in rats sacrificed 4 hr after oral administration of 100 mg mancozeb or zineb/kg body weight. Mancozeb inhibited p-nitroanisole O-dealkylase and aniline hydroxylase to a greater degree than zineb, whereas the inhibition of aminopyrine N-demethylase by the two fungicides was quantitatively comparable. Interestingly, aryl hydrocarbon hydroxylase (AHH) which exhibited maximum inhibition with mancozeb, remained unaffected following zineb administration. The time-course and dose-dependence of MFO inhibition examined 1 and 4 hr after a single oral dose of 100 mg or 250 mg zineb/kg was expressed as a dose-dependent decline in the rate of xenobiotic biotransformation at 4 hr. In vitro interaction of zineb with MFO resulted in slightly greater inhibition of aminopyrine, p-nitroanisole and aniline while AHH exhibited more pronounced decrease with mancozeb. The magnitude of inhibition of aminopyrine N-demethylase and AHH was independent of the time of preincubation of fungicides with the enzyme. Kinetic studies indicated the non-competitive nature of AHH inhibition. Chronic oral treatment with mancozeb and zineb at a dose of 250 mg/kg for 4 weeks did not modify xenobiotic biotransformations except for a slight induction of aminopyrine N-demethylase by mancozeb.     

Quantitative determination of anti-tumor agent bis(4-fluorobenzyl)trisulfide, fluorapacin and its pharmaceutical preparation by high-performance liquid chromatography

A simple isocratic and stability-indicating HPLC method was developed and validated for the quantitative determination of anti-tumor agent fluorapacin and its pharmaceutical preparation. A Spherisorb ODS II C(18) (250 mm x 4.6 mm, 5 microm) column was eluted with a mobile phase consisting of acetonitrile/water (85:15, v/v). The analyses were performed at 40+/-1 degrees C with a flow rate of 1.0 mL/min and UV detection at 218 nm. The calibration curve was linear over a concentration range of 160-240 microg/mL with the correlation coefficient of 0.9997. The LOD and LOQ were determined to be 1.4 and 7.0 ng/mL, respectively. Average recoveries were 98.27% and 100.40% for fluorapacin API and its drug product with corresponding relative standard deviations (R.S.D.) of 0.41% and 0.30%, respectively. Good repeatability (precision and intermediate precision), accuracy and tolerability were obtained with R.S.D. of <1.0%. This specific and reliable method has been successfully applied for quality control of fluorapacin API and drug product.     

Stability-indicating methods for determination of vincamine in presence of its degradation product

Three different stability indicating assay methods are developed and validated for determination of vincamine in the presence of its degradation product (vincaminic acid). The first method is based on the derivative ratio zero crossing spectrophotometric technique using 0.1 N hydrochloric acid as a solvent. In the second method, measurements are based on spectro-densitometric technique using high performance thin-layer chromatography (HPTLC) plates with a developing system consisting of methanol-chloroform-ethyl acetate (2:1:1, v/v/v). The third method depends on high-performance liquid chromatography (HPLC). Separation of vincamine from vincaminic acid using Lichrocart RP-18 column (250 mm x 4.6 mm i.d.) with a mobile phase consisting of acetonitrile-ammonium carbonate (0.01 M) (7:3, v/v) is achieved. The methods showed high sensitivity with good linearity over the concentration ranges of 12 to 48 microg ml-1, 3 to 17 microg/spot, and 2 to 20 microg ml-1 for derivative spectrophotometry, spectro-densitometry and HPLC methods, respectively. The developed methods were successfully applied to the analysis of pharmaceutical formulations containing vincamine with excellent recoveries.     

Simultaneous isocratic HPLC determination of vigabatrin and gabapentin in human plasma by dansyl derivatization

A rapid and low-cost assay for simultaneous vigabatrin (VGA) and gabapentin (GBP) determination is described that can be performed with simple HPLC instrumentation. The method involves derivatization of the primary amine group of VGA and GBP with dansyl chloride followed by isocratic separation (column: microBondapak C-18, 10 microm, 300 x 3.9 mm; mobile phase: 50 mmol/L NaH(2)PO(4) in 40% acetonitrile) at 50 degrees C and fluorometric detection (excitation and emission wavelength: 318 and 510 nm, respectively) of the fluorescent product, which is stable for at least 7 days. Correlation coefficients of the calibration curves are >0.999 with a lower limit of detection of 0.3 microg/mL. Between- and within-run coefficients of variation are below 4.5%, and assay time is 15 minutes. This method may be used for therapeutic drug monitoring in the case of GBP and to control patient compliance in the case of VGA.     

Optimized procedure for lamotrigine analysis in serum by high-performance liquid chromatography without interferences from other frequently coadministered anticonvulsants

The authors have developed a simple isocratic high-pressure liquid chromatographic (HPLC) assay for the simultaneous determination of lamotrigine and other frequently coadministered antiepileptic drugs in serum samples. Lamotrigine extraction was performed on a reversed-phase Oasis HBL preparation column. The eluates containing butalbital as internal standard were separated with a 7-microm Chromsystems C18 250 x 4.0 mm I.D. reversed-phase column at a temperature of 40 degrees C using a mobile phase consisting of pH 3.8 phosphate-acetonitrile buffer (55:45, v/v), at a flow rate of 0.8 mL/min. Ultraviolet detection was carried out at 210 nm. Measurement of the peak:height ratio allowed quantitative determination of the samples. The method was linear over a concentration range of 0.2 to 20 microg/mL for lamotrigine. Recovery was >90%. Within-day and between-day coefficients of variation ranged from 1.8% to 6.7%. The mean lamotrigine concentration was 8.01 +/- 5.63 microg/mL. After studying sera from 130 patients treated with lamotrigine the authors confirmed that associated antiepileptic therapy affected the serum lamotrigine levels, which were significantly higher in patients under valproic acid treatment.     

Determination of 5-fluorouracil and its prodrug tegafur in plasma and tissue by high-performance liquid chromatography in a single injection: validation for application in clinical pharmacokinetic studies

Tegafur, a prodrug of 5-fluorouracil (5-FU), is an oral fluorouracil antitumor drug used for the management of adenocarcinomas. It has an efficacy similar to that of intravenous 5-FU, with potential advantages in terms of convenience and quality of life for the patient and cost-effectiveness as compared with intravenous chemotherapy. The authors developed a high-performance liquid chromatography (HPLC) assay for the determination of tissue or plasma tegafur and 5-FU concentration in a single step extraction and a single HPLC injection. The retention times of 5-FU and tegafur were 5 and 16.5 minutes, respectively, and the internal standard retention times were 11.5 and 17.5 minutes for 5-bromouracil (5-BU) and beta-hydroxyethyltheophylline, respectively. The limit of quantification was 0.0125 microg/mL for 5-FU and 0.05 microg/mL for tegafur. The assay had good recovery (96.5% +/- 9.45% and 97.5% +/- 7.89% for 5-FU in plasma and tissue, respectively, and 88.5% +/- 12.17% and 104.9% +/- 8.77% for tegafur in plasma and tissue, respectively). Precision was good: the within-day and between-day standard deviation of the mean (RSD) for 5-FU (0.0125-5 microg/mL) and tegafur (0.5-150 microg/mL) was always <8%. The authors conclude that the method described here is ideally suited for the therapeutic monitoring of 5-FU and tegafur.     

A new, rapid, fully automated method for determination of fluconazole in serum by column-switching liquid chromatography

A sensitive and rapid HPLC assay for the determination of fluconazole in serum is described. HPLC-integrated sample preparation allows direct injection of serum samples without any pretreatment. The in-line extraction technique is carried out by automatically switching from the extraction column (Lichrospher ADS C8) to the analytic column (Nucleosil C18). After 6 minutes the matrix passes the extraction column, and the retained analyte is quantitatively transferred to the analytic column, where separation by isocratic HPLC is performed. The extraction eluent is sodium dihydrogen phosphate buffer, pH 5.0 (50 mM), and the analytic eluent is acetonitrile/sodium dihydrogen phosphate buffer, pH 5.0 (50 mM) (26.8/73.2, vol/vol). Fluconazole is detected according to its absorption maximum at 210 nm. The lower limit of quantification (LLOQ) is 0.65 microg/mL, the limit of detection (LOD) is 0.2 microg/mL, and the quantification range is 0.65-23.3 microg/mL. The assay was precise with a between-run coefficient of variation of < or = 5.59%. The within-run accuracy was 99.8% and 103.4%, and the between-run accuracy was 99.2% and 99.7%, respectively, for the concentrations 23.3 microg/mL and 1.3 microg/mL. The recovery was 78%. The described procedure allows sample cleanup and determination within 20 minutes, thereby facilitating drug monitoring in clinical routine. The method was applied successfully.     

Toxicological analysis of chlorhexidine in human serum using HPLC on a polymer-coated ODS column

A simple and reliable high-performance liquid chromatographic (HPLC) method for analyzing chlorhexidine in human serum was developed. After the addition of an internal standard, levomepromazine, 0.2 mL serum was deproteinized with 10% perchloric acid. The acidic supernatant was neutralized with 1M potassium carbonate solution, and the insoluble salt was removed by centrifugation. An aliquot of the supernatant was applied to HPLC with UV detection (260 nm). HPLC separation was achieved on a polymer-coated ODS column equilibrated with acetonitrile/water containing 0.05% trifluoroacetic acid, 0.05% heptafluorobutyric acid, and 0.1% triethylamine (40:60, v/v). The calibration curve was linear in the concentration range from 0.05 to 50.0 microg/mL, and the lower limit of detection was 0.05 microg/mL. The accuracy and precision of the method were evaluated at concentrations of 0.5 microg/mL and 5.0 microg/mL. The coefficients of variation ranged from 4.0 to 4.5%. The concentration of chlorhexidine in the serum of a patient who died after a suspected intravenous injection of chlorhexidine gluconate was determined.     

Quantitative determination and sampling of azathioprine residues for cleaning validation in production area

Cleaning validation is an integral part of current good manufacturing practices in any pharmaceutical industry. Nowadays, azathioprine and several other pharmacologically potent pharmaceuticals are manufactured in same production area. Carefully designed cleaning validation and its evaluation can ensure that residues of azathioprine will not carry over and cross contaminate the subsequent product. The aim of this study was to validate simple analytical method for verification of residual azathioprine in equipments used in the production area and to confirm efficiency of cleaning procedure. The HPLC method was validated on a LC system using Nova-Pak C18 (3.9 mm x 150 mm, 4 microm) and methanol-water-acetic acid (20:80:1, v/v/v) as mobile phase at a flow rate of 1.0 mL min(-1). UV detection was made at 280 nm. The calibration curve was linear over a concentration range from 2.0 to 22.0 microg mL(-1) with a correlation coefficient of 0.9998. The detection limit (DL) and quantitation limit (QL) were 0.09 and 0.29 microg mL(-1), respectively. The intra-day and inter-day precision expressed as relative standard deviation (R.S.D.) were below 2.0%. The mean recovery of method was 99.19%. The mean extraction-recovery from manufacturing equipments was 83.5%. The developed UV spectrophotometric method could only be used as limit method to qualify or reject cleaning procedure in production area. Nevertheless, the simplicity of spectrophotometric method makes it useful for routine analysis of azathioprine residues on cleaned surface and as an alternative to proposed HPLC method.     

In vitro studies of cellular and molecular developmental toxicity of adjuvants, herbicides, and fungicides commonly used in Red River Valley, Minnesota

Recent epidemiologic studies showed increased frequency of birth defects in pesticide applicators and general population of the Red River Valley, Minnesota. These studies further indicated that this crop growing area used more chlorophenoxy herbicides and fungicides than elsewhere in Minnesota. Based on frequency of use and known biology, certain herbicides, pesticide additives, fungicides, and mycotoxins are suspect agents. To define whether these agents affect developmental endpoints in vitro, 16 selected agrochemicals were examined using the MCF-7 breast cancer cell line. In the flow cytometric assay, cell proliferation in this estrogen-responsive cell line indicates xenobiotic-mediated estrogenic effects. Cell viability, morphology, ploidy, and apoptosis were incorporated in this assay. Data showed that the adjuvants X-77 and Activate Plus induced significant cell proliferation at 0.1 and 1 microg/ml. The commercial-grade herbicides 2,4-D LV4 and 2,4-D amine induced cell proliferation at 1 and 10 microg/ml. The reagent-grade 2,4-D products failed to induce proliferation over the same concentration range, suggesting that other ingredients in the commercial products, presumably adjuvants, could be a factor in these results. The fungicides triphenyltin and mancozeb induced apoptosis at concentrations of 4.1 microg/ml (10(-5) M) and 50 microg/ml, respectively. Triphenyltin also induced aneuploidy (C2/M arrest) at 0.41 microg/ml (10(-6) M). Data provide a mechanistic step to understanding human reproductive and developmental effects in populations exposed to these agrochemicals, and initiative to focusing limited resources for future in vivo animal developmental toxicity studies.     

The ethylene bisdithiocarbamate fungicides mancozeb and nabam alter essential metal levels in liver and kidney and glutathione enzyme activity in liver of Sprague-Dawley rats

Mancozeb is a fungicide of the ethylene bisdithiocarbamate (EBDC) class complexed to the metals manganese and zinc. Nabam is the sodium salt of the EBDC backbone. The purpose of this study was to determine if these EBDC compounds alter essential metal homeostasis and glutathione status in Sprague-Dawley rats. Our findings indicate EBDCs caused accumulation of copper in kidneys, but not liver. EBDC compounds also increased glutathione reductase activity in liver, but not kidneys, whereas only mancozeb increased glutathione peroxidase activity in the liver. Mancozeb and nabam increased total glutathione in liver, but only mancozeb increased total glutathione in the kidney. Neither mancozeb nor nabam altered glutathione ratio in either liver or kidney compared to control. Our data suggest that the EBDC backbone of mancozeb, and not the zinc or manganese moieties, is responsible for changes in glutathione status and alteration of essential metal homeostasis in rat liver and kidney.     

Development and validation of RP HPLC method to determine nandrolone phenylpropionate in different pharmaceutical formulations

This study describes development and subsequent validation of a reversed phase high performance liquid chromatographic (RP-HPLC) method for the estimation of nandrolone phenylpropionate, an anabolic steroid, in bulk drug, in conventional parenteral dosage formulation and in prepared nanoparticle dosage form. The chromatographic system consisted of a Luna Phenomenex, CN (250 mm x 4.6 mm, 5 microm) column, an isocratic mobile phase comprising 10 mM phosphate buffer and acetonitrile (50:50, v/v) and UV detection at 240 nm. Nandrolone phenylpropionate was eluted about 6.3 min with no interfering peaks of excipients used for the preparation of dosage forms. The method was linear over the range from 0.050 to 25 microg/mL in raw drug (r2 = 0.9994). The intra-day and inter-day precision values were in the range of 0.219-0.609% and 0.441-0.875%, respectively. Limits of detection and quantitation were 0.010 microg/mL and 0.050 microg/mL, respectively. The results were validated according to International Conference on Harmonization (ICH) guidelines in parenteral and prepared nanoparticle formulation. The validated HPLC method is simple, sensitive, precise, accurate and reproducible.     

Simultaneous determination of zonisamide, carbamazepine and carbamazepine-10,11-epoxide in infant serum by high-performance liquid chromatography

This study developed a simple method for the simultaneous determination of zonisamide (ZNS), carbamazepine (CBZ) and its active metabolite, carbamazepine-10,11-epoxide (CBZE) in infant serum using reversed-phase high-performance liquid chromatograph (HPLC). The method involves a single-step protein precipitation procedure that uses no solid-phase or liquid-liquid extraction. The HPLC separation was carried out on a Cadenza CD-C18 column (3 microm, 4.6 mm x 150 mm) with potassium phosphate buffer (pH 4.6; 25 mM)-methanol-acetonitrile (65:20:15 (v/v/v)) as a mobile phase at a 1.0 ml/min flow rate: ZNS was detectable using a UV detector at 235 nm, and both CBZ and CBZE were at 215 nm. The quantification limits were established in accordance with each therapeutic range at 2.5 microg/ml for ZNS, 0.5 microg/ml for CBZ, and 0.25 microg/ml for CBZE. The respective coefficients of variation were 1.3-6.0% and 2.2-7.7% for the intra- and inter-assay.     

A specific and rapid HPLC assay for the determination of cefroxadine in human plasma and its application to pharmacokinetic study in Korean

A specific and rapid high performance liquid chromatographic (HPLC) method with UV detection (254 nm) was developed for the determination of cefroxadine in human plasma. The sample extraction was performed by a simple procedure, vortexing and centrifugation of sample following addition of 60% trichloroacetic acid. Cephalexin was used as an internal standard (I.S.). The HPLC analysis was carried out on a Capcell Pak C18 analytical column with a mobile phase of 50 mM ammonium formate buffer/pH 3.5 and acetonitrile (90:10, v/v). No interference was observed near the peaks of cefroxadine and I.S. The calibration curve was linear over the range of 0.5-40 microg/mL and the lower limit of quantification (LLOQ) was 0.5 microg/mL. The method was validated with excellent sensitivity, accuracy, precision and stability. This assay was successfully applied to determine the pharmacokinetic parameters of cefroxadine in Korean healthy volunteers after an oral administration of two 250 mg cefroxadine capsules. As a result, the plasma half-life was 1.00+/-0.26 h and the mean AUC(0-6 h) was 46.25+/-6.41microgh/mL. The maximum plasma concentration (C(max)) of 17.62+/-4.87 microg/mL reached 1.44+/-0.39 h after administration.     

Analysis of captopril and hydrochlorothiazide combination tablet formulations by liquid chromatography

A reverse-phase, high-performance liquid chromatographic (HPLC) procedure was developed for the simultaneous assay of captopril and hydrochlorothiazide in a combination tablet formulation. Gradient elution was used to quantify these two drugs, as well as the oxidized form of captopril, the disulfide. Tablets were extracted with methanol and, after centrifugation, were chromatographed. Initially, a methanol-0.05% aqueous phosphoric acid (25:75, v/v) solution was pumped at 2 mL/min into a phenyl column. After 8 min, the flow rate was increased to 4.5 mL/min and the methanol content of the mobile phase was increased to 45% to elute the disulfide. Detection was at 210 nm. Linearity and repeatability of all constituents were satisfactory. The hydrolytic degradation product of hydrochlorothiazide, 4-amino-6-chloro-1,3-benzene disulfonamide (also called the disulfonamide ), could be resolved in test solutions but was not visible in chromatograms of tablets carried through the gradient procedure even after storage at elevated temperatures for prolonged time periods prior to assay. The method can be automated.     

HPLC法测定大鼠血浆中黄芩苷的浓度

目的建立测定大鼠血浆中黄芩苷浓度的HPLC方法。方法用甲醇-乙腈沉淀血浆蛋白,将上清液吹干后残渣再溶解后进行测定;色谱柱采用DiamonsilC18柱,以甲醇-0.2%磷酸(60:40,v/v)为流动相,流速为1.0mL/min,检测波长为280nm,选取卡马西平为内标物。结果黄芩苷在0.25～10.0μg/mL范围内呈良好线性关系(r=0.9990),日内、日间RSD均小于5%;平均相对回收率与平均提取回收率分别为98.4%和69.3%。结论该方法简单快速,适用于黄芩苷的血药浓度测定及药动学研究。     

Determination of plasma and brain levels of isotretinoin in mice following single oral dose by high-performance liquid chromatography

An isocratic reversed-phase high-performance liquid chromatographic method was established and validated according to FDA's Guidance for Industry, "Bioanalytical Method Validation", for the determination of isotretinoin in plasma and brain tissue from mice following single and multiple oral doses of Accutane. Plasma sample preparation included deproteination with acetonitrile-perchloric acid followed by centrifugation. Brain tissue was homogenized and extracted with acetonitrile-perchloric acid followed by centrifugation. The supernatants were analyzed by high-performance liquid chromatography (HPLC). Benz[alpha]anthrancene-7,12-dione was used as the internal standard. Chromatographic separation was achieved on a C18 column using an acetonitrile-aqueous 0.5% acetic acid (85:15, v/v) elution. The average extraction efficiency was >95% for plasma and >82% for brain. The lower limit of quantification was 30 ng/mL for plasma and was 30 ng/0.1g for brain tissue, respectively. The linear range for plasma was 30-600 ng/mL, and 15-300 ng/0.1g for brain. Maximum concentrations of isotretinoin in both plasma and brain were observed at 1h after single oral dosing (25 mg/kg). The maximum concentrations in plasma and brain were 2.36 microg/mL and 0.34 microg/g, respectively. The mean area under curve (AUC) in plasma was 6.13 microg h/mL. The mean eliminate half-life in plasma was estimated as 46 min.     

Simultaneous determination of paracetamol and methocarbamol in tablets by ratio spectra derivative spectrophotometry and LC

The application of the ratio spectra derivative spectrophotometry and high-performance liquid chromatography (HPLC) to the simultaneous determination of paracetamol (PAR) and methocarbamol (MET) in combined pharmaceutical tablets is presented. The spectrophotometric procedure is based on the use of the first derivative of the ratio spectra obtained by dividing the absorbtion spectrum of the binary mixtures by a standard spectrum of one of the compounds. The first derivative amplitudes were measured at 243.0 and 230.3 nm for the assay of PAR and MET, respectively. Calibration graphs were established for 2-30 microg ml for PAR and 2-36 microg/ml for MET in binary mixture. The detection limits for PAR and MET were found 0.097 and 0.079 microg/ml, respectively; while the quantification limits were 0.573 microg/ml for PAR and 1.717 microg/ml for MET. For the HPLC procedure, a reversed-phase column with a mobile phase of methanol-water (60:40, v/v), was used to separate both compounds with a detection of 274.0 nm. Linearity was obtained in the concentration range of 2 300 and 1.5-375 microg/ml for PAR and MET, respectively. The detection and quantification limits were found to be 0.42 and 1.4 microg/ml for PAR and 0.36 and 1.2 microg/ml for MET, respectively. The relative standard deviations were found to be less than 0.52%, indicating reasonable repeatibility of both methods. The proposed methods were successfully applied to the determination of these drugs in commercial tablets.