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.     

Automated Solid Phase Extraction and Hplc Analysis of Ibuprofen in Plasma

Abstract

Ibuprofen is a non-steroidal anti-inflammatory drug, widely used in arthritis and other disorders. We describe a high pressure liquid chromatographic (HPLC) method for the analysis of ibuprofen in plasma, using an automated solid phase extraction technique (the Varian AASP^R). In this method ibuprofen was extracted from 0.5 ml of plasma by application to a C₂ extraction cartridge followed by “on line” elution with the HPLC mobile phase (55% acetonitrile / 45% 0.02 H phosphate buffer; pH 3.0), at a flow rate of 1.5 ml/min. The analytical column was a Nucleosil C₁₈ column and the fluorescence detector was set at 253 nm (excitation wavelength) and 300 nm (emission wavelength). Chromatography was complete in less than 10 mins and the limit of detection was 1.3 /μg/ml. The method is linear through the range of 1.0 to 100.0 /μg/ml with a mean correlation coefficient of 0.9964. Absolute recovery of ibuprofen from the spiked plasma samples ranged from 77.8% to 86.5%. The method was shown to be precise within 11% C.V. and accurate to within 8% over the concentration range studied.     

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.     

High Performance Liquid Chromatographic Determination of Pentamidine in Plasma

Abstract

A high performance liquid chromatographic method for quantitating pentamidine in plasma has been developed. Sample clean-up involved precipitating plasma with acetonitrile containing the internal standard, hexamidine. The supernatant was passed through a C₈ Bond Elut column and eluted with a methanolic solution of sodium 1-heptanesulfonate. The eluate was then analyzed on an Altex C₈ column with a mobile phase consisting of 45% CH₈CN, 0.02% detramethylammonium chloride and 0.1% H₃PO₄. Using fluorescence detection (EX: 275 nm and EM: 340 nm), the detection limit was 1.25 ng/ml for 0.5 ml of plasma. The coefficients of variation for interday and intraday were around 10%.     

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.     

A Simple and Sensitive HPLC Method for Antipyrine in Plasma

Abstract

A rapid, simple and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of antipyrine in small volume (50 μl) of plasma samples. Aminopyrine was employed as the internal standard. The sample preparation is a direct plasma protein precipitation procedure so is less tedious and rapid. The assay employs a column packed with a C₁₈ reversed-phase material (5 μm Nucleosil) with an isocratic mixture of acetonitrile and water (25:75, v/v) as the mobile phase. The eluant was detected at 254 nm. The assay achieves the level of sensitivity (0.5 μg/ml) and accuracy required to obtain meaningful data about the single-dose pharmacokinetics of antipyrine in guinea pig and rat. The method gave high reproducibility with coefficients of variation less than 5%.     

High Performance Liquid Chromatographic Determination of Amiodarone in Plasma and Tissues

Abstract

A simple and rapid high-performance liquid chromatographic (HPLC) method for the determination of amiodarone (AD) in plasma and tissues was developed. The method involved deproteinization of plasma or homogenized tissue with acetonitrile containing an internal standard (N-Cetylpyridinium chloride) followed by reversed phase chromatography using μ bondapack C₁₈ column (10μm) with a mobile phase consisting of acetonitrile - methanol - sodium dihydrogen phosphate buffer (70:10:20%, v/v), the pH adjusted to 4.0 and pumped at flow rate of 1.0 ml/min. The column effluent was monitored at 242 nm. A linear relationship was obtained between peak height ratios (drug to internal standard) versus drug levels over the concentration range of 50–750 ng/ml. The detection limit of AD in plasma and tissues by this method was 20 ng/ml.     

Liquid Chromatography‐Electrospray Ionization Mass Spectrometric Method for Determination of Gliclazide in Human Plasma

Abstract

A rapid, sensitive, and specific liquid chromatography‐electrospray ionization mass spectrometric (LC‐ESI‐MS) method has been developed for quantification of gliclazide in human plasma. The analyte and tolbutamide (internal standard, I.S.) were extracted from plasma samples with n‐hexane–dichloromethane (1:1, v/v) and analyzed on a C₁₈ column. The chromatographic separation was achieved within 4.0 min by using methanol–0.5% formic acid (80:20, v/v) as mobile phase and the flow rate was 1.0 mL/min. Ion signals m/z 324.0 and 271.0 for gliclazide and internal standard were measured in the positive mode, respectively. The method was linear within the range of 2.5–2000 ng/mL. The lower limit of quantification (LLOQ) was 2.5 ng/mL. The intra‐ and inter‐day precisions were lower than 2.8% in terms of relative standard deviation (RSD). The inter‐day relative error (RE) as determined from quality control samples (QCs) ranged from ?1.93% to 1.85%. This validated method was successfully applied for the evaluation of pharmacokinetic profiles of gliclazide modified‐release tablets in 20 healthy volunteers.     

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.     

A Novel Method for the Quantitation of Warfarin and its Metabolites in Plasma

Abstract

A procedure for the rapid, quantitative recovery of warfarin and its metabolites (diastereoisomeric alcohol, 4′-, 6-, 7-, 8-benzylic-hydroxywarfarin and dehydrowarfarin) from plasma with Sep-Pak C₁₈ cartridges has been developed. A solution of warfarin and its metabolites in plasma was acidified with NH₄OAc buffer (pH 4.85), adsorbed on the Sep-Pak C₁₈ resin, washed free of polar constituents and eluted with methanol. Dilution of the eluate with buffer followed by gradient high performance liquid chromatography permitted accurate quantitation of the desired compounds when detected at 313 nm. The recovery of warfarin and each metabolite was greater than 95% over an investigated range of 0.5–10.0 μg/ml of plasma and the limit of quantitation by the assay was 0.1 μg/ml of plasma. For more rapid quantitation of warfarin, without simultaneous analysis of metabolites, the chromatographic parameters were modified so that elution of warfarin occurred within 13 minutes, and the sensitivity of the assay increased to 0.03 μg of warfarin/ml of plasma. The quantitative recovery of warfarin and its metabolites coupled with the chromatographic versatility of the method make it ideally suited for either detailed pharmacokinetic studies or routine plasma analysis of warfarin.     

Determination of Ferulic Acid in Rat Plasma by Liquid Chromatography–Tandem Mass Spectrometry Method: Application to a Pharmacokinetic Study

Abstract

A rapid, sensitive, and specific liquid chromatography–tandem mass spectrometric (HPLC-MS/MS) method has been developed for quantification of ferulic acid in rat plasma. The analyte and docetaxel (internal standard) were extracted from plasma samples with diethyl ether and analyzed on a C₁₈ column. The chromatographic separation was achieved within 3.5 min by using acetonitrile-water as the mobile phase and the flow rate was 0.2 mL · min^?1. The method was linear within the range of 0.5 ? 800 ng · mL^?1. The lower limit of quantification (LLOQ) was 0.5 ng · mL^?1. Finally, the method is successfully applied for the pharmacokinetic study of ferulic acid in rats following intravenous administration.     

Development and validation of a reversed-phase HPLC method with post-column iodine-azide reaction for the determination of thioguanine

A high-performance liquid chromatographic method of reversed-phase with a post-column iodineazide reaction has been developed and validated for the determination of thioguanine. Isocratic elution was performed on a column of C₁₈ using acetonitrile- water-sodium azide solution (1.5%; pH 6.5) 16: 34: 50 (v/v/v) as a mobile phase with flow-rate of 0.5 mL/min. Monitoring of unreacted iodine in post-column iodine-azide reaction induced by thioguanine resulted in its detection at 350 nm. The method applied to thioguanine was linear within the scope of values 8–100 nM (r ² > 0.9988). The relative standard deviation (RSD < 4.2%) and the recovery (>96%) prove that the intra-day precision and the accuracy were satisfactory. The lower limits of detection (LLD) and quantification (LLQ) of thioguanine were established at the levels of 6 and 8 nM, respectively. The elaborated method was validated and applied to thioguanine determination in tablets.     

Size Exclusion Chromatographic Determination of PEG 3350 in Human Plasma and Urine

Abstract

A size exclusion chromatographic method is reported that quantitated PEG 3350 in human plasma and urine to a concentration of 10 μg/ml in plasma, or urine. The chromatographic system consisted of a 500 A gel permeation analytical column, a differential refractometer detector, and chloroform as the eluting solvent. Organic extraction was used as an initial separation technique. PEG 400 was used as the internal standard for PEG 3350, and showed similar extraction properties. Spiked plasma standards yielded standard calibration curves with correlation coefficients of greater than 0.99, and relative standard deviations (n=3) of 2.19% (500 μg/ml) and 6.97% (20 μg/ml). The analytical technique was used to estimate the elimination rate constant of PEG 3350 from 5 normal human subjects after oral administration of 240 grams of PEG 3350. K_E was found to be 0.1079 hr^?1 0.03781^?1 hr (mean s.d.) using the Sigma-minus data analysis method on total urines collected from the 5 subjects at varying intervals.     

Determination of Pyridostigmine in Human Plasma by High-Performance Liquid Chromatography

Abstract

An easy to perform, specific, reproducible and sensitive high performance liquid chromatographic (HPLC) method to measure pyridostigmine concentration in human plasma was developed and validated. Sample clean-up consists of ion-pair extraction into dichloromethane in the presence of neostigmine as internal standard, followed by back extraction into an aqueous phase. Mean recovery of 110% (with a standard deviation of 10%) was determined for concentrations of 5 – 100 ng/ml. Chromatography on a 125·4 mm CN-propyl column using a mobile phase composed of 10% acetonitrile in 3.5×10^?4M NaH₂PO₄ and UV detection at 270 nm, yields clean chromatograms without any interferences from endogenous plasma components. Using 1 ml plasma samples the method has a limit of detection (LD) of 3 ng/ml, with %CV (precision) and bias (accuracy) ≥ 10% for concentrations in the range of 0–100 ng/ml. The method is being used in human pharmacokinetic studies of oral dosage forms of pyridostigmine.     

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.     

A Simple High-performance Liquid Chromatographic Assay for Loracarbef in Human Plasma

Abstract

A simple, rapid and reproducible high-performance liquid chromatographic (HPLC) method for the determination of loracarbef in human plasma has been developed and evaluated. Plasma protein was precipitated with ammonium sulfate. The drug and the internal standard (Cefetamet) were eluted from a μ-bondapak C-18 column with a mobile phase consisting of acetonitrile:methanol:water:glacial acetic acid (2.5:17.5:79.2:0.8%, v/v). The column eluent was monitored at 265 nm. Quantification was achieved by the measurement of the peak-height ratio of the analyte to the internal standard and the limit of quantification for loracarbef in plasma is 0.5 ug/ml. The within-day coefficient of variation (CV) ranged from 2.28% to 3.67%, and between-day CV from 2.38% to 5.59% at three different concentrations. The absolute recoveries ranged from 91.1% to 93.88%, and the relative recoveries from 93.4% to 108% at three different concentrations. Preliminary stability tests showed that loracarbef is stable for at least 5-weeks in human plasma after freezing. The method is applied for the determination of the pharmacokinetic parameters of loracarbef after oral administration to 2 beagle dogs.     

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.     

Determination of Arteether in Plasma Using a Simple and Rapid High-Performance Liquid Chromatographic Assay

Abstract

A simple and rapid high-performance liquid chromatographic (HPLC) assay for the determination of the antimalarial drug arteether in plasma was developed and validated in this report. Perchloric acid was used in this method as a plasma protein precipitant and to attain an acidic medium suitable for the decomposition of arteether to a derivative possessing UV absorption. This derivative and the internal standard (progesterone) were separated from the plasma on a 10 μm μ-Bondapack C₁₈ reversed-phase column at ambient temperature with a mobile phase composed of acetonitrile:water (60:40 v/v) and at a flow rate of 1.5 ml/min. The effluent was monitored at 254 nm with a UV detector. Linear relation between drug concentrations and peak height ratios of arteether derivative to the internal standard was achieved in the range of 0.25-10 μg/ml arteether with a detection limit of 50 ng/ml arteether in plasma. The within-day and between-days precisions were evaluated using 3 different concentrations of arteether. The values of the coefficients of variation were 1.35-1.68% and 1.65-2.82% for within-day and between-day, respectively. This method was applied to determine some pharmacokinetic parameters of arteether after intramuscular injection of 50 mg/kg arteether oily solution to rabbits.     

Rapid Quantitation of Verapamil in Plasma by High-Performance Liquid Chromatography

Abstract

A simple and sensitive liquid chromatographic assay procedure using a fluorescence detector for the quantitative determination of verapamil in plasma without extraction was developed. After precipitating the protein with acetonitrile, the resulting supernatant liquid was injected onto the column for analysis. Chromatographic separation was achieved on C₁₈ reversed phase column and the eluting solvent was the isocratic mixture of methanol, acetonitrile and pH 3.0 glycine buffer (1:4:5). With this mobile phase the drug and its internal standard were well separated from the interference of the plasma sample. The average recovery of verapamil from 3 replicate samples of different concentration (100–600 ng/mL) were 95.5 ± 5.68%. The minimum amount of verapamil detectable by this method was 40 ng/mL of sample. The elimination half-life (β-phase) of this drug in rabbits was found to be 3.7 hours.     

A Sensitive High Performance Liquid Chromatographic Determination of Clopamide in Human Plasma

Abstract

A simple and sensitive high-performance liquid chromatographic method for quantitation of clopamide in human plasma has been developed. the assay uses a reversed-phase C18 microbore column (2 mm I.D. × 100 mm) packed with 5 μm ODS Hypersil. the chromatographic separation was achieved by using an isocratic mobile phase comprising acetonitrile-10 mM phosphate buffer pH 4 (17:83, v/v) at a flow rate of 0.5 ml/min. the eluant was monitored by a UV detector operating at 241 nm. the assay was based on an organic extraction before chromatographic separation. to 1 ml plasma sample, 100 μl of the internal standard, methylparaben (300 ng/ml), and 8 ml of diethyl ether were added. the samples were shaken and centrifuged, the organic layer was then transferred to a tapered centrifuge tube and evaporated to dryness. the residue was reconstituted and injected onto the HPLC column. the inter-and intra-assay coefficients of variation were found to be less than 10%. the lowest limit of detection for clopamide in plasma was 5 ng/ml. the method is sensitive, specific and allows for routine analysis in the pharmacokinetic studies.