High Performance Liquid Chromatographic Analysis of Tolmetin,Indomethacin and Sulindac in Plasma

Abstract

Isocratic and gradient reversed phase high-performance liquid chromatographic (HPLC) methods for the quantitation of tolmetin, indomethacin, and sulindac and their respective metabolites in plasma were developed. Only the determination of the parent drugs was possible using the isocratic technique. Specific, simultaneous determination of each drug and its respective metabolites was achieved using the gradient technique. The effect of pH and ionic concentration of the mobile phase on retention time was studied. Statistical analysis demonstrated excellent precision and linearity over the following ranges: 1–40, 0.1–3, and 0.1–3 ug/ml plasma for tolmetin, indomethacin, and sulindac respectively. Both methods have been applied to the analysis of patient samples.     

A Simple Isocratic HPLC Method for the Determination of Metoclopramide in Plasma and Urine

Abstract

Metoclopramide concentrations in plasma and urine were determined by high performance liquid chromatography using a cyanopropylsilane column and UV detection. The mobile phase consisted of 0.03M sodium acetate (pH 7.4) and acetonitrile. The plasma samples were extracted with dichloromethane after pH adjustment. Urine proteins were precipitated with acetonitrile. The reproducibility and precision of the methods were demonstrated by the analysis of samples containing 5 – 200 ng/ml plasma and 0.25 – 200 ug/ml urine.

The glucuronide and sulfate conjugates of metoclopramide were also quantitated after differential acid hydrolysis of urine samples. The conditions for acid hydrolysis were studied. The methods have been applied to the analysis of plasma and urine samples obtained from human volunteers.     

Spectrophotometric Determination of Narcotine

Abstract

Narcotine reacts with potassium iodate and antimony potassium tartarate to give a yellowish orange colour having maximum absorbance at 430–440 nm. The reaction is specific for narcotine, having 1 ug/10 ml as visual limit of identification and provides a basis for a new spectropho-tometric determination of pure narcotine and in the presence of other drugs. The maximum tolerable limits of these are reported.     

A New Simplified Microassay for the Quantitation of Theophylline in Serum by High-Performance Liquid Chromatography

Abstract

An improved, rapid and simplified HPLC method is presented for the quantitation of theophylline in serum. The internal standard, beta-hydroxypropyl theophylline (20 ul of a 100 ug/ml solution) is added to 50 ul of serum. Serum proteins are precipitated by the addition of 30 ul of 20% trichloroacetic acid solution. The mobile phase consists of a sodium acetate buffer with 8% acetonitrile. Chromatogram run time is 8 minutes. The sensitivity limit is 0.10 ug/ml. This method is interference free from the major metabolites of theophylline and other drugs commonly coadministered with theophylline.     

Simultaneous Determination of CGS 10787B and Its Major Metabolite (CGS 12094) in Plasma by High Performance Liquid Chromatography

Abstract

A method for the simultaneous determination of CGS 10787B and its major, metabolite (CGS 12094) in plasma is described. The two compounds, and the internal standard (dichlorinated analog), are extracted from acidified plasma with ethyl acetate, taken to dryness, and reconstituted in chromatographic mobile phase. The analytes are determined automatically by high performance liquid chromatography in the reversed-phase mode as paired ions, using [N(Bu)₄]⁺ as the counterion. The separation of the compounds is achieved on a 3u C-8 column, with detection at 254 nm.

Recovery and reproducibility assessments indicate good accuracy and precision over the range of 1.0 to 250 ug/ml for CGS 10787B and 1.0 to 100 ug/ml for CGS 12094.

The method has a limit of detection of 0.2 ug/ml for both compounds, and has been shown to be adequate for studying the disposition kinetics of CGS 10787B.     

Simultaneous Determination of Phenylbutazone,Flunixin, Indomethacin and 4 Methylaminoantipyrene with Novel use of an Ion-Pairing Step-Gradient HPLC Methodology

Abstract

An inexpensive step-gradient elution of the above nonsteroidal anti-inflammatory drugs (NSAIDs) using HPLC is described. The method involves: ion pairing the acid NSAIDs with tetrabutylammonium hydroxide (TBA) at pH 7.2, a stepwise change between two mobile phases differing only in solvent strength, and a “dilute and shoot” plasma sample preparation.

The limit of sensitivity of the assay if 0.5 meg/ml with a signal to noise ratio of 3 to 1 and linearity to 20 mg/ml.     

Determination of Doxorubicin,Daunorubicin and some of their Metabolites in Mouse Plasma by High-Performance Reversed-Phase Liquid Chromatography with Amperometric Detection

Abstract

A selective and sensitive reversed-phase liquid chromatographic method with electrochemical detection for the analysis of doxorubicin, daunorubicin and some of their metabolites in plasma is reported. A mobile phase consisting of acetonitrile-phosphate buffer solution-tetrahydrofuran (25–71,5–3,5) flowing at 1 ml/min through a Lichrocart RP 18 column was employed. The influence of various parameters on the separation (solvent composition, pH, tetrahydrofuran content) has been examined. An extraction of anthracyclines from plasma was performed using chloroform-ethanol mixture (4: 1) with high extraction efficiency; reproducible results were attained by working with a 1 M phosphate buffer which ensured a real buffering of the plasma samples. The sensitivity of amperometric detection makes this method suitable for analyzing small amounts of the parent drugs and their metabolites. The precision was better than 4% in the range 0.2 to 5 μg/ml plasma.     

A Stability-Indicating Hplc Analysis of Famotidine and Its Application to Kinetic Studies

Abstract

A stability-indicating HPLC analytical method has been developed for the determination of the H₂-receptor antagonist, famotidine in the presence of its degradation products. the method utilizes reversed phase chromatography with UV detection and internal calibration techniques. the mobile phase was comprised of 84% ammonium acetate buffer (pH 2.9) and 16% acetonitrile and pumped at a flow rate of 1.5 ml/min. Quantitation was performed by measuring the peak height ratio of drug to internal standard (salicylic acid). the limit of famotidine detection was determined to be 10 ng (0.4 ug/ml) with a signal to noise ratio of 3:1. Within day coefficient of variation of the method was 2.22% (2.5 μg/ml) and 0.82% (10 μg/ml). Between day coefficient of variation based on the slopes of daily prepared standard curves was 4.70%. the developed method was used to determine the drug content of famotidine tablets. Further, it was used to investigate the kinetics of degradation of the drug in an acidic solution.     

Atomic Emission Spectrometric Determination of Antazoline,Hydralazine, Amiloride,Thiamine and Quinine Based on Formation of Ion Associates with Ammonium Reineckate.

Abstract

Ion-associate complexes of Antazoline HC1 (I), Hydralazine HC1 (II), Amiloride HC1 (III), Thiamine HC1 (IV) and Quinine sulphate (V) with ammonium reineckate were precipitated and their solubilities were studied as a function of pH, ionic strength and temperature. Saturated solutions of each ion-associate under the optimum precipitation conditions were prepared and the metal ion-content in the supernatent was determined. The solubility products were thus elucidated at different temperatures. A new accurate and precise method using direct coupled plasma-atomic emission spectrometry for the determination of the investigated drugs in pure solutions and in pharmaceutical preparations is described. The drugs can be determined by the present method in the ranges 0.3-3.0, 0.19-1.96, 0.3-3.0, 0.33-3.37 and 0.78-7.82 mg/25 ml solutions of I, II, III, IV and V, respectively.     

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.     

Measurement of Nicotine in Plasma by High-Performance Liquid Chromatography

Abstract

A new procedure has been developed to measure nicotine in blood plasma by high-performance liquid chromatography (HPLC). Nicotine is extracted from plasma by elution with cholorform. Final determination is achieved by isocratic HPLC with ultraviolet detection. Twenty microliters of plasma extract is deluted over a silica column at a flow rate of 1.0 ml/min with a dioxane:isopropanol:NH₄OH (80:3.0:0.4) mobile phase. The procedure is sensitive to 0.05 ug of nicotine per ml of plasma and is linear within the range of 0.05 to 10.0 ug/ml of plasma. When a known amount of nicotine was added to plasma, the concentration of nicotine measured averaged 99.9 + 3.9 (S.D.)% of the known concentration. The within-sample coefficient of variation was 3.9%     

Specific Differential Spectrophotdmetric Determination of Ascorbic Acid in Plants Using Ascorbate Oxidase

Abstract

A specific enzymic-differential spectrophatometric method is described and evaluated for the determination of ascorbic acid in vegetables and medicinal plants. It is based on the absorbance measurement at 593 nm of the complex of ferrous ion with 2,4,6-tris (pyridyl)-S-triazine, which is produced by reduction of ferric ion by ascorbic acid, versus a blank sample treated with ascorbate oxidase. The absorbance difference is linearly related to ascorbic acid from 10 to 100 ug/ml. A standard deviation of ±0.5 ug/ml (n=5) and a mean recovery of 101.47. (98.3–103.3%.) from spiked plant extracts, were found. The method was used to determine ascorbic acid in various plants of the Greek flora. Acacia cvanophvlla was found to be the richest source tested for ascorbic acid (89.6 mq/100 q of leaves). The method is very simple and can be used in routine analysis.     

An HPLC Method for the Determination of Diltiazem and Desacetyldiltiazem in Human Plasma

Abstract

A high performance liquid chromatographic method is presented for the determination of diltiazem and its metabolite desacetyldiltiazem in human plasma. Diltiazem and desacetyldiltiazem are extracted from plasma basified with 0.5M dibasic sodium phosphate (pH 7.4) using 1% 2-propanol in n-hexane containing diazepam as an internal standard. A reversed phase cyanopropylsilane column was used with a mobile phase of 45% acetonitrile and 55% 0.05M acetate buffer (pH 4.0). The minimum detectable limit was 2ng/ml of plasma. The effect of the pH, molarity, and percent acetonitrile of the mobile phase on the capacity factor was studied. Possible interferences from other drugs administered concurrently are presented.     

Small angle neutron scattering studies on micellar systems part 2. Mixed systems of ammonium decanoate and ammonium perfluoro-octanoate

Mixtures of ammonium decanoate (AmDec) and ammonium perfluoro-octanoate (APFO), in ammonium chloride: ammonium hydroxide buffer at pH 8.8 and an ionic strength of 0.1, were examined by small angle neutron scattering with varying proportions of the two surface active agents. The results indicated that mixed micelles were formed and it is suggested from analysis of the data that these were cylindrical in shape at APFO: AmDec ratios of 21, 11 and 12. At 21 and 11 the micelles were found to contain more APFO than expected on the basis of ideal mixing of the surface active agents in the micelle. At a ratio of 19 the micelles were found to be spherical but larger than those formed from AmDec alone. A speculative model is proposed for the mixed micelle which still, however, allows for segregation between hydrocarbon and fluorocarbon chains within the micelle.     

Analysis of Adenosine and Other Adenine Compounds in Patients With Immunodeficiency Diseases

Abstract

Adenine compounds can be measured in picomole amounts using liquid chromatography of the fluorescent 1, N⁶-etheno derivatives. The limit of detection for the etheno derivatives in tissue extracts, however, is tissue-dependent due to interference by nucleotides and fluorescent components which are normally present. Prior to derivatization nucleotides were partially removed from extracts of lymphocytes and erythrocytes by treatment with Dowex AG1-X2 anion exchange resin. Samples were analyzed using either a Partisil PXS 10/25 SCX column eluted with 100 mM NH₄H₂PO₄, pH 4.5, at a flow rate of 2 ml/min; or using two μBondapak/C₁₈ reversed-phase columns eluted with 5 mM KH₂PO,₄:25% methanol (V/V) pH 7.5, at a flow rate of 1 ml/min. Adenosine was found to be 0.07 nmole/ml in normal adult human plasma. The urine of a child with severe combined immunodeficiency disease associated with absence of adenosine deaminase contained a normal amount of adenosine (5–6 nmole/ml), but contained a high level (～60 nmole/ml) of deoxyadenosine. Deoxyadenosine was not detected (<0.01 nmole/ml) in normal adult urine. Because of its sensitivity and selectivity, this method of analysis should be applicable to studies of the physiological roles of adenine compounds.     

High-Performance Liquid Chromatography of MDL-035 in the Plasma of Rats,Dogs and Humans

Abstract

A sensitive and reproducible high performance liquid chromatographic method was set up for the assay of MDL-035, a new non-steroidal, nonacidic analgesic antiinflammatory agent, in the plasma of rats, dogs and humans. Plasma samples (0.5ml) containing flurazepam as the internal standard, were diluted and extracted with ethyl ether. After centrifugation, the organic phase was taken to dryness, the residue was redissolved and injected into an RP-2 column. The elution was made in isocratic conditions using a CH.CN/phosphate buffer solution as mobile phase. The UV detection was made at 320 nm. The method was validated for the concentration range from 0.05 to 10 ug/ml, and applied to pharmacokinetic studies. A typical plasma concentration-time profile in the rat after an oral administration is here presented.     

Simultaneous Determination of Tinidazole and Furazolidone in Suspension by HPTLC and HPLC

Abstract

High performance thin layer chromatographic (HPTLC) and high performance liquid chromatographic (HPLC) methods were developed for the simultaneous determination of Tinidazole and Furazolidone in suspension.

In the HPTLC method the separation of Tinidazole and Furazolidone was carried out on silica gel 60F₂₅₄ HPTLC glass plate using chloroform:methanol:ammonia (9:1:0.1 v/v) as a mobile phase. Rf values obtained were 0.63 and 0.79 for Furazolidone and Tinidazole respectively. Densitometric evaluation was done at 335 nm. Linearity was obtained within the concentration range 10–50 μg/ml and 3.5–17.5 μg/ml for Tinidazole and Furazolidone respectively.

The second method is based on high performance liquid chromatography on a reversed phase column (μ Bondapak C₁₈) using a mobile phase comprised of water: acetonitrile: triethylamine (80:20:0.1 v/v) adjusted to pH = 3.0 with dil. phosphoric acid. Retention times were 5.24 and 7.82 min for Tinidazole and Furazolidone respectively at a flow rate of 1.5 ml/min. Detection was done at 335 nm. Linearity was obtained within the concentration range 30–180 μg/ml and 10.5–63 μg/ml for Tinidazole and Furazolidone resp.     

Spectrophotofluorimetric Determination of Trace Amounts of Aluminium with 2,2′-Dihydroxy-3,3′-dicarboxy-1, 1′-Dinaphthylmethane (Pamoic Acid)

Abstract

Fluorescence properties of 2,2′-dihydroxy-3,3′-dicarboxy -1,1′-dinaphthylmethane (pamoic acid) and of its aluminium 1:1 complex are described. The fluorescence of the latter, as measured from a water-dioxane (3:7, v/v) solution, at pH 5.05–5.20, at room temperature, is used as a basis for a new method of aluminium determination at submicrogram level. Limit of quantification of the proposed procedure is 0.028 ng.ml^?1, range of applicability goes up to 1.7 ug.ml^?1, RSD is 1% at 0.47 ug.ml^?1 level (concentrations, given refer to HCl solution which is measured) Tolerance ratios for several interferent metal ions are given. A comparison is made with Kirkbright's method which uses 3-hydroxy-2-naphthoic acid as complexing agent: stability with time of the readings and freedom from Fe(III) interference are the main advantages of the proposed procedure. The new method is applied to the aluminium determination in atmospheric aerosol samples.     

An HPLC Method for the Determination of Verapamil and Norverapamil in Human Plasma

Abstract

A high performance liquid chromatographic method is presented for the determination of verapamil and its metabolite norverapamil in human plasma. Verapamil and norverapamil are extracted from plasma basified with 0.5M dibasic sodium phosphate (pH 9.5) using ethyl acetate containing trimipramine as an internal standard. A reverse-phase cyanopropylsilane column was used with a mobile phase of 65% acetonitrile and 35% 0.02M acetate buffer (pH 7.0). The minimum detectable limit was 2 ng/ml of plasma. The effect of the pH, molarity, and percent acetonitrile of the mobile phase on the capacity factor was studied. Possible interferences from other drugs administered concurrently are presented.     

Flow Injection Analysis of Carboxylic Acid Drugs Using Cationic Dyes and On-Line Ion-Pair Extraction

Abstract

A flow injection system containing an on-line ion-pair extractor has been designed for the analysis of carboxylic acid drugs using either spectrophotometric detection with gentian violet as counterion or fluorescence detection with acridine orange. Salicylic acid, valproic acid, and ibuprofen were selected as model drugs. A mobile phase of 90:10 aqueous pH 7 phosphate buffer-absolute methanol was pumped through the system at 1 ml/min. A chloroform solution of the cationic dye was pumped into the mobile phase at 1.25 ml/min and the chloroform layer containing the dye-drug ion-pair separated prior to detection. Peak height and absorbance were linear for salicylic acid, valproic acid, and ibuprofen in the 0.5–10, 5–50, and 1–10 μg/ml ranges, respectively. Peak height and fluorescence were linear for salicylic acid, valproic acid, and ibuprofen in the 0.13–5, 2.5–50, and 0.5–20 μg/ml ranges, respectively. Accuracy and precision for the spectrophotometric assays were in the 2–6% and 3.3–6.6% ranges, respectively, and in the 0.3–4% and 1.3–5.4% ranges, respectively, for the fluorescence assays. Peak height and absorbance were also shown to be linear in the 16–500 μg/ml range for prostaglandin PGF_2α with accuracy and precision of 1–3% and 5–6%, respectively, for spiked samples. Commercial dosage forms containing valproic acid and ibuprofen were assayed by the spectrophotometric assay and found to be within acceptable USP limits. Spiked ibuprofen samples at the 5 and 10 μg/ml level were assayed using an octadecylsilane column inserted into the flow injection system. One to two percent accuracy and 2.5–5% precision were obtained for the drug using the FIA-column system.