Extractionless HPLC Method for the Determination of Naproxen in Human Plasma and Urine

Abstract

A simple and rapid (extractionless) high-performance liquid chromatographic method with ultraviolet detection, at 278 nm, is described for the determination of naproxen in human plasma and urine. Niflumic acid is used as internal standard. The chromatographic system consists of a reversed-phase C18-Spherisorb column with acetonitrile/0.1 M sodium acetate (35:65 v/v, pH 6.14) as the mobile phase. The retention time is 3.0 min for naproxen and 3.8 min for niflumic acid. The total run time is 5 min and the typical assay time is 10 min. The method is sufficiently sensitive for biopharmaceutical studies, after the oral administration of a single sustained release dose.     

A Rapid Assay of Suramin in Plasma

Abstract

The polysulfonated napthylurea suramin is currently undergoing extensive clinical trials. A narrow therapeutic window requires frequent dose adjustment to minimize toxicity. A simple, rapid and reproducible assay was developed for measuring suramin levels in plasma. Plasma samples are injected directly onto the column and eluted isocratically with acetonitrile and an ion pairing reagent. Retention time is approximately two minutes, recovery greater than 95% throughout the range of 50–300 ug/ml, with sensitivities to concentrations of 5 ug. A small sample size (0.5 ul) gave similar results allowing assays of finger-stick, samples. Equal accuracy was abtaiaedusingtrypanblueasaninterndlstandaril. careful d-, maWabxy for safe ncaninistmtion of the agent, can be acccilplisbed rapidly and accurately.     

Determination of Saccharin in Plasma and Urine by High Performance Liquid Chromatography

Abstract

A sensitive and specific high performance liquid chromatographic (HPLC) assay for the determination of saccharin in plasma and urine was developed. Saccharin is extracted into diethyl ether at acid pH, evaporated, and reconstituted prior to instrumental analysis. Overall recovery of saccharin is 86.9 + 8.6% and the sensitivity limits of detection is 0.15 μg per ml of plasma or urine using a fluorescence detector. The sensitivity limit in plasma can be extended to 20 ng per ml by use of a 2 ml assay volume and detector attenuation. The assay was used for the determination of saccharin in plasma and urine of rats following oral doses of 5 mg/kg.     

HPLC Assay for S-2-(3-Aminopropylamino)ethyl Phosphorothioate (WR 2721) in Plasma

Abstract

A specific HPLC assay has been developed for determination of the radioprotective drug WR 2721. The method is based on precolumn derivatization of plasma with fluorescamine, separation with a C-18 cartridge and detection by fluorescence. An external standard was used for calibration, and values were adjusted based upon recovery of added ¹⁴C-labeled WR 2721. WR 2721 had a retention time of about 13 minutes using a mobile phase of acetonitrile/water (22:78), 0.01 M in dibutylammonium phosphate, at a flow rate of 2 mL/min. Sensitivity of the assay was characterized to 2 μg/mL, and detector response was linear over the range of 2 to 1100 μg/mL. The assay requires 90 μL of plasma and has a total chromatography time of about 45 minutes. 2-(3-Aminopropylamino)ethanethiol (WR 1065) and bis- [2- (3-aminopropylamino)ethyl]disulfide (WR 33278), metabolites of the drug, and a variety of primary amines were shown not to interfere with the assay. Suitability of this assay for pharmacokinetic studies was demonstrated in preliminary experiments with a beagle dog.     

Rapid High Performance Liquid Chromatographic Determination of Ampicillin in Human Urine

Abstract

A rapid, sensitive and specific HPLC assay for the determination of ampicillin in human urine is developed.

Ampicillin was directly measured in human urine at 225 nm using a reversed phase column (Synchropack RP-P) and a mobile phase composed of (1:9 methanol-sodium acetate solution, 0.01 M, pH 4). The analysis required no longer than 10 min. Linear correlation between the peak height ratio of ampicillin to cefoxitin sodium (internal standard) and ampicillin concentration in urine over the range 10–100 μg ml^?1 was obtained. The developed method proved to be advantageous as it monitors ampicillin level in urine. Moreover, the urinary excretion of ampicillin in human subjects after an oral administration of 500 mg ampicillin capsules was established using the proposed method.     

Simultaneous Determination of Salicylic Acid and Salicyluric Acid in Plasma and Urine by High Pressure Liquid Chromatography

Abstract

A sensitive, rapid, and specific high pressure liquid chromatographic (HPLC) assay was developed for the determination of salicylic (SA) and salicyluric (SU) acids in plasma and urine. The compounds are extracted into ethyl ether at acid pH, evaporated, and reconstituted prior to instrumental separation. Overall recovery of both compounds is 90 ± 5%, and the sensitivity limits are 150 ng of SU and 300 ng SA per ml of biological fluid. The assay was used for the determination of both compounds in plasma and urine of man following oral doses of 40 mg/kg of sodium salicylate.     

Quantification of Suramin by Reverse-Phase Ion-Pairing High-Performance Liquid Chromatography

Abstract

A specific and sensitive method has been developed for the separation and quantification of suramin and trypan blue (internal standard) in human plasma. Plasma samples were extracted by centrifugation after the addition of ion-pairing reagent (tetra-butylammonium phosphate, TRAP) and methanol. Extracts were injected directly onto a reverse-phase ion-pairing HPLC system with 5 mM TBAP in the mobile phase. There was nearly 100% extraction efficiency after 3 cumulative extracts of each sample. The limit of quantitation was 0.5 μg/ml at a detection wavelength of 313 nm. Analysis of 3 post-therapy samples from a patient with AIDS was used to determine a plasma half-life for suramin of at least 3 weeks.     

High Performance Liquid Chromatographic Separation of Estradiol-17 α and -17/β in Biological Fluids; Application to Plasma,Milk and Urine of Cows

Abstract

A rapid HPLC technique was developed to separate estradiol epimers. In order to improve the sensitivity of the detection, a radioitmmunoassay was used.

Estrone, estradiol-17α and estradiol-17β were separated within 20 min using 10 ml of chloroform: acetone (90:10), as the mobile phase. The efficiency of the technique was assessed with ₃ steroids and the assay of collected fractions with antlsera specific to each estrogen. Using a non-specific radioimmunoassay, profiles of endogenous estrogens in different biological fluids (blood plasma, milk, urine) were obtained.

The efficiency of HPLC as a separation method and the high sensitivity of radioimmunoassay as a detector allows us to obtain profiles of estrogens from biological samples where steroid concentration is below lOOpg/ml.     

High-Performance Liquid Chromatographic Determination of Acetazolamide in Human Plasma

Abstract

A simple, rapid and specific HPLC method has been developed to determine acetazolamide concentrations in human plasma. The assay procedure requires only 250 μl of sample with direct injection of the organic supernatant after protein precipitation with acetonitrile. Chlorothiazide was used as an internal standard. A reversed-phase C₁₈ μBondapak column was employed for the chromatographic separation. The eluent was monitored at 265 nm using a UV variable wavelength detector. The retention times for acetazolamide (ACZ) and chlorothiazide (CTZ) were 6 and 8 min respectively. A linear relationship (r).995) was obtained over the 1-20 μg/ml concentration range. The limit of sensitivity for ACZ was 0.5 μg/ml, with greater than 85% recovery of ACZ and internal standard. The method was applied to human plasma samples obtained after administration of a 250 mg acetazolamide tablet.     

An Ion-Pairing High-Pressure Liquid Chromatography Assay for the Determination of Cefoperazone in Plasma and Urine

Abstract

A high-performance liquid chromatographic assay for cefoperazone (cfp) in plasma and urine is described. For analysis, the internal standard ticarcillin (ticar) is solvated in acetonitrile, which is then added to plasma or urine. The supernatant is drawn off of the resulting protein precipitate and injected directly onto the reverse-phase C₁₈ column, with detection at 254 nm. The mobile phase is composed of phosphate-acetonitrile-tetramethylammonium chloride (TMA). Coefficients of variation for reproducibility were less than 9% for extra-low, low, medium, and high controls. Limits of detection were 0.5 μG/mL for plasma and 1 μG/mL for urine. No interference from other cephalosporins or other antibiotics was found. This high-pressure liquid chromatographic ion-pairing assay is simple, accurate, inexpensive, and requires no extraction.     

High-Performance Liquid Chromatographic Determination of Atenolol in Tablets

Abstract

A reversed phase high-performance liquid chromatographic method was developed for the determination of atenolol in four oral 100 mg atenolol preparations.

An aliquot of the sample is dissolved in a mobile phase consisting of 0.0612 M potassium hydrogen phosphate - isopropanol-tetrahydrofuran (84:10:6) v/v). The pH was adjusted to 6.7 with phosphate buffer. Nicotinamide was used as internal standard and chromatographed on a Pinkerton column ISRP (GFF-S5–80) 5 μm, 150 × 4.6 mm i.d. The applied column is convenient for the assay at least 90 samples of atenolol without degrading column performance. The detection was performed at 272 nm. The retention time for atenolol was 5.07 min.

The proposed HPLC method was found to be suitable for the rapid and precise routine analysis of atenolol in tablets.     

Determination of Ibafloxacin,A New Quinolone Antibacterial,in Human and Dog Plasma and Urine by High Performance Liquid Chromatography

Abstract

A simple, selective and sensitive high performance liquid chromatographic method has been developed for quantifying plasma and urine ibafloxacin levels in humans and dogs.

Sample pretreatment is done by incorporation of an internal standard (IS) followed by a single step chloroform extraction. Samples are then chromatographed by reverse phase chromatography with UV detection. The lowest quantifiable concentration is 0.1 μg ibafloxacin/mL with a 1 mL sample. The assay was linear over the range of 0.1–50 μg/mL.     

Simultaneous Gas Chromatographic Determination of Diazapam and its Major Metabolites in Human Plasma,Urine, and Saliva

Abstract

A glc analysis method was developed for the simultaneous determination of diazepam (I), and its major metabolites N-desmethyldiazepam (II), oxazepam (III), and hydroxydiazepam (IV) in human plasma, urine, and saliva. Medazepam (V) was used as the internal standard to control extraction efficiency and permit precise and accurate determination of I-IV. Extraction and glc analysis of physiological fluid samples in triplicate required only 40 minutes using the developed method. Three human subjects were given either 20 or 5 mg of I via oral administration and serial specimens of blood, urine, and saliva collected. All samples were analyzed using the developed assay method. Results indicate that the method could be applied to pharmacokinetic studies of I where plasma, urine, and/or saliva is to be monitored.     

Reverse Phase HPLC Determination OF 5,6-Dihydro-5-azacytidine in Biological Fluids

Abstract

A sensitive and specific reverse phase HPLC method which allows measurement of the new antitumor agent 5,6-dihydro-5-azacytidine (DHAC) in biological fluids at concentrations as low as 50 ng/ml (2 × 10^?7 M) has been developed. After addition of 5′-chloro-5′-deoxy-5,6-dihydro-5-azacytidine as an internal standard, sequential ultrafiltration, boronate gel affinity chromatography and cation exchange chromatography are employed to isolate DHAC from plasma or urine. DHAC is then reacted with N,N-dimethylformamide diethylacetal to form a dimethylaminomethylene derivative with enhanced UV detectability (λmax = 264 nm, log ε = 4.3) and better retention on a reverse phase column. Isocratic separation is then accomplished on a fully loaded and end-capped ODS column with 0.050 M formic acid in 20% acetonitrile/water. This assay has been used to determine the plasma pharmacokinetics of DHAC in rats given a single i.v. bolus dose of 50 mg/kg. Analysis of the drug in human plasma indicates that this method is suitable for determining DHAC disposition and pharmacokinetics in human subjects.     

A Rapid Liquid Chromatographic Method for Determination of Flecainide in Human Blood Plasma Using Ultraviolet Detection

Abstract

A liquid chromatographic method for the assay of the antiarrhythmic drug flecainide in plasma has been developed. The method is rapid, simple and with sufficient detection sensitivity to render it suitable for therapeutic drug monitoring. Flecainide and added internal standard, a non-fluorinated analogue, were extracted by a single ether extraction from alkalinized plasma followed by a back-extraction of the ether with dilute phosphoric acid. A portion of the acid extract was then applied directly to a 30 cm ODS column eluting isocratically with 30% acetonitrile in water containing 0.01M dibutylamine phosphate. Monitoring was by ultraviolet detection at 214 nm and the total run time was 8 min. This method is specific and can quantitate plasma levels to less than 30 ng/ml (free base) from 0.5 ml of plasma without interference from antiarrhythmic drugs commonly used in therapy.     

Gas Chromatographic Determination of Hexamethylene Bisacetamide in Plasma and Urine

Abstract

A rapid, simple and specific gas chromatographic method has been developed to measure the differentiating agent hexamethylene bisacetamide (HMBA) in biological samples. Addition of a homolog as an internal standard, ultrafiltration and then direct packed column GC-FID analysis of the ultrafiltrate gives a detection limit of less than 50 μg/ml (0.25 mM) for HMBA in plasma or urine. Ultrafiltration is quantitative and assay precision is better than 4.3% for the 1–5 mM range. This method has been applied to determine the bolus dose pharmacokinetics and disposition of HMBA in a single small animal such as a rat. The developed assay should be suitable for therapeutic monitoring of human patients undergoing HMBA treatment.     

An Improved HPLC Method for the Determination of Furosemide in Plasma and Urine

Abstract

A sensitive HPLC method with minimal sample preparation and good reproducibility for the determination of furosemide in plasma and urine is described. Acidified plasma samples were extracted using CH₂Cl₂ containing desmethylnaproxen as internal standard (IS). Fresh urine samples were incubated with β-gluc-uronidase for 15 minutes and then treated with CH₃CN containing IS.

Chromatography was performed on a C18 column with 10 mcl sample injection, Mobile phases were: a) for plasma: 0.01 M NaH₂PO₄, pH 3.5 - CH₃OH (65:35), and b) for urine: acetic acid, pH 3.5 - CHS3OH (60:40) at 3 ml/min and fluorescence detection at Ex 235/Em 389 nm. The plasma standard curve was linear from 0.01 to 15.0 mcg/ml and the urine from 0.5 to 200.0 mcg/ml. The within run CV's were 3,2% at 0.74 mcg/ml plasma and 2.0% at 10.7 mcg/ml urine. Recovery from plasma was 69.9% at 2.0 mcg/ml and 98.6% from urine at 5.0 mcg/ml. The stability of furosemide and its glucuronide were studied. Both methods have been applied to the analysis of plasma and urine samples obtained from human volunteers.     

Determination of Ciprofloxacin (Bay o 9867) in Biological Fluids by High-Performance Liquid Chromatography

Abstract

A high-performance liquid chromatographic method for the analyses of ciprofloxacin (BAY o 9867) (1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylic acid hydrochloride) in human serum, plasma and urine samples is described. Diluted serum, plasma, and urine samples are injected onto a RP-18 column without prior extraction or clean-up procedure. Ciprofloxacin is separated from the ballast by an eluent consisting of an 0.025M H₃PO₄ solution adjusted to pH=3 with tetrabutylammonium hydroxide and acetonitrile.

Ciprofloxacin is detected fluorimetrically giving a detection limit of 8ng/ml in plasma and serum and of 50ng/ml in urine. A statistical evaluation of the assay showed acceptable accuracy and precision for 10 to 500ng of BAY o 9867 per ml in serum and plasma and for 50ng to 600ng of BAY o 9867 per ml of diluted urine specimens. This method was used to monitor the concentrations of BAY o 9867 in serum, plasma and urine of volunteers after oral administration of ciprofloxacin.     

A Rapid Qualitative and Quantitative Method of Assaying Histamine in Small Plasma Volume

Abstract

A modified method for a qualitative and quantitative determination of histamine in small plasma volume (≤300 μl) was developed. According to this method, blood samples containing methylhistamine, the internal standard, are centrifuged to collect plasma. These plasma samples which contain underivatized histamine are injected into a Dionex BioLC System coupled with a pulsed amperometric detector. Histamine and methylhistamine are separated through a C-18 Zorbax ODS 4.5mm ID ± 25cm (5 microns) column. Histamine is quantitated by comparing histamine peak height with that of known quantity of the internal standard. The sensitivity of the method is 0.03 pmols. The peak heights were found to be linearly related to histamine concentrations providing a quantitative means of assaying histamine in biological samples. The retention time of histamine was 6 min in contrast to that of methlyhistamine which was 10 min.     

HPLC Measurement of Cyclosporine in Blood Plasma and Urine and Simultaneous Measurement of its Four Metabolites in Blood

Abstract

A high performance liquid chromatographic assay has been developed for the estimation of cyclosporine and its four major metabolites in blood and for cyclosporine alone in plasma and urine samples. This assay employs a rapid and very reproducible solid-liquid extraction system. Isocratic chromatographic conditions allow the simultaneous measurement of cyclosporine and its four major metabolites in blood. The method is linear up to 2500 ng/ml and the minimum quantifiable limit for cyclosporine is 30 ng/ml, when 1 ml of sample is analyzed.