High Performance Liquid Chromatographic Analysis of Desipramine in Human Plasma Using a Microbore Column Technique

Abstract

A reversed-phase, isocratic HPLC method has been developed for the quantitation of desipramine in human plasma. the method involved the use of cloimpramine as an internal standard. the chromatographic separation was accomplished with a mobile phase comprising acetonitrile-aqueous solution (60:40. v/v) containing 10 mM disodium hydrogenphosphate and 80 mM sodium dodecyl sulfate adjusted to pH 2. the mobile phase was pumped at a flow rate of 0.5 ml/min. the column used was a microbore column (2 mm I.D. × 100 mm) packed with a C18 reversed-phase material (5μm ODS Hypersil). Plasma samples were extracted at basic pH with diethyl ether followed by back-extraction into 0.1 N sulfuric acid. Using UV detection at 250 nm, the lower limit of sensitivity was 10 ng/ml. the inter- and intra-assay coefficients of variation were found to be less than 10%. the assay procedure was applied to a long term oral dosing study in patients to monitor the plasma concentration of desipramine.     

Reversed-phase high-performance liquid chromatographic analysis of atenolol enantiomers in plasma after chiral derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate.

A sensitive high-performance liquid chromatographic method for the determination of the enantiomers of atenolol in rat plasma has been developed. Racemic atenolol and practolol (internal standard) were extracted from alkalinized plasma (pH 12) into dichloromethane containing 3% (v/v) heptafluoro-1-butanol, and the organic layer was evaporated. The samples were derivatized with (+)-1-(9-fluorenyl)ethyl chloroformate at pH 8.5 for 30 min. After removal of excess reagent, the diastereomers were extracted into dichloromethane. The diastereomers were separated on a Microspher C18 column (3 microns) with a mobile phase of acetonitrile-sodium acetate buffer (0.01 M, pH 7) (50:50, v/v) at a flow-rate of 0.8 ml/min. Fluorescence detection (lambda ex = 227 nm, lambda em = 310 nm) was used. When 100 microliters of plasma were used, the quantitation limit was 10 ng/ml for the atenolol enantiomers. The assay was applied to measure concentrations of atenolol enantiomers in plasma after intravenous administration of racemic atenolol to rats.     

Simultaneous determination of propranolol and 4-hydroxypropranolol in plasma by mass fragmentography.

A quantitative method for the simultaneous determination of propranolol and its active metabolite 4-hydroxypropranolol in human plasma is described. Plasma samples are extracted at pH 9.6 with ethyl acetate after the addition of sodium bisulphite and the internal standard oxprenolol. The extracts are derivatized with trifluoroacetic anhydride before separation on a gas chromatograph--mass spectrometer. Detection and quantitation of the trifluoroacetyl derivatives are made by single-ion monitoring. The minimum detectable concentration of propranolol is 1 ng/ml and of 4-hydroxypropranolol 5 ng/ml using 1-ml plasma samples. No interferences from normal plasma constituents or from drugs commonly prescribed together with propranolol were detected.     

Rapid and sensitive gas chromatographic determination of diacetylmorphine and its metabolite monoacetylmorphine in blood using a nitrogen detector.

A quantitative gas chromatographic method for the determination of plasma concentrations of diacetylmorphine and its metabolite monacetylmorphine using an alkali flane detector (nitrogen detector) is described. Plasma samples (pH 9.0) containing ethylmorphine acetate as internal standard are extracted with benzene. The dried benzene extracts are analysed as their corresponding acetylated derivatives following treatment with trifluoroacetic anhydride-benzent (1:5). The nitrogen detector permits quantitation of narcotic levels down to 100ng/ml with detection as low as 20 ng/ml. The higher sensitivity and selectivity of the nitrogen detector are compared to those obtained in flame ionization detection. Species differences in the rate of conversion of diacetylmorphine to monacetylmorphine in vitro in blood are also presented.     

Determination of the catechol-O-methyltransferase inhibitor Ro 40-7592 in human plasma by high-performance liquid chromatography with coulometric detection.

A sensitive and specific high-performance liquid chromatographic method has been developed to measure the catechol-O-methyl-transferase (COMT) inhibitor 3,4-dihydroxy-4'-methyl-5-nitrobenzophenone (Ro 40-7592) in human plasma. The compound and the internal standard were extracted from plasma at pH 2 with n-butyl chloride-ethyl acetate (95:5, v/v). The extract was chromatographed on a reversed-phase column (Hypersil ODS, 5 microns) using a mixture of phosphate buffer (0.05 M, pH 2), methanol and tetrahydrofuran (45:55:5, v/v/v) as the mobile phase. Long-retained components were removed from the system by means of a simple column-switching system. Quantification of the catechol-O-methyltransferase inhibitor was performed by means of coulometric detection (0.1 V). The limit of quantification was about 1 ng/ml, using a 1-ml specimen of plasma. The recovery from human plasma was greater than 88%. The mean inter-assay precision was 5.3% in the range 2.5-1000 ng/ml. Linearity of the standard curve was obtained in the concentration range 2.5-500 ng/ml. The catechol-O-methyltransferase inhibitor was stable in human plasma when stored for six months at -20 degrees C and for 24 h at room temperature. The practicability of the new method was demonstrated by the analysis of more than 400 plasma samples from a tolerance study performed in human volunteers.     

Simultaneous Determination of Flunixin,Phenylbutazone, Oxyphenbutazone and γ-Hydroxyphenylbutazone in Equine Plasma by High-Performance Liquid Chromatography: With Application to Pharmacokinetics

Abstract

A high performance liquid chromatographic method was developed for the simultaneous determination of flunixin, phenylbutazone, oxyphenbutazone and γ-hydroxyphenylbutazone in equine plasma. Samples of plasma or sera were deproteinated by addition of acetonitrile containing the internal standard naproxen. The concentration step consisted of taking an aliquot of deproteinated plasma, evaporating under nitrogen to dryness and redissolving in mobile phase. The extracts were chromatographed on a Spherisorb 5 μm ODS column using an isocratic mobile phase of methanol (30% v/v), acetonitrile (20% v/v) and pH 3.0 1% acetate buffer (50% v/v) at a flow rate of 1.2 ml/min using naproxen as the internal standard. The detection limit for flunixin, phenylbutazone, oxyphenbutazone and γ-hydroxyphenylbutazone was 50 ng/ml.

The developed chromatographic method was applied to the determination of equine nonsteroidal anti-inflammatory treatment. Plasma samples from clinically treated horses administered flunixin and phenylbutazone simultaneously are reported. Effect of different anticoagulants used in sampling is reported.     

Quantitative determination of famotidine in human maternal plasma,umbilical cord plasma and urine using high‐performance liquid chromatography–mass spectrometry

Liquid chromatography with electrospray ionization mass spectrometry for the quantitative determination of famotidine in human urine, maternal and umbilical cord plasma was developed and validated. The plasma samples were alkalized with ammonium hydroxide and extracted twice with ethyl acetate. The extraction recovery of famotidine in maternal and umbilical cord plasma ranged from 53 to 64% and 72 to 79%, respectively. Urine samples were directly diluted with the initial mobile phase then injected into the HPLC system. Chromatographic separation of famotidine was achieved by using a Phenomenex Synergi? Hydro‐RP? column with a gradient elution of acetonitrile and 10 mm ammonium acetate aqueous solution (pH 8.3, adjusted with ammonium hydroxide). Mass spectrometric detection of famotidine was set in the positive mode and used a selected ion monitoring method. Carbon‐13‐labeled famotidine was used as internal standard. The calibration curves were linear (r² > 0.99) in the concentration ranges of 0.631–252 ng/mL for umbilical and maternal plasma samples and 0.075–30.0 µg/mL for urine samples. The relative deviation of method was <14% for intra‐ and inter‐day assays, and the accuracy ranged between 93 and 110%. The matrix effect of famotidine in human urine, maternal and umbilical cord plasma was less than 17%. Copyright © 2013 John Wiley & Sons, Ltd.     

Simple High-Performance Liquid Chromatographic Method for the Analysis of Quinine in Human Plasma without Extraction

Abstract

A simple, rapid and sensitive assay, capable of quantitating quinine (Q) in human plasma samples is reported. The assay uses a reversed-phase C18 HPLC column packed with 5 μ ODS Hypersil. The chromatographic separation was accomplished with an isocratic mobile phase comprising acetonitrile-aqueous phosphate buffer pH 2 (50:50, v/v) containing 25 mM sodium dodecyl sulfate and 3 mM tetrabutylammonium bromide at a flow rate of 0.5 ml/min. The eluant was monitored by a fluorescence detector (excitation wavelength at 350 nm and emission wavelength at 450 nm). The assay was based on a simple plasma protein precipitation technique. To 200 μ of plasma sample, 400 μ of internal standard (cinchocaine 30 μ/ml in methanol) was added. After brief vortexing and centrifugation, the clear supernatant was 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 Q in plasma was 18 ng/ml.     

Quantitative analysis of EO9 (apaziquone) and its metabolite EO5a in human plasma by high-performance liquid chromatography under basic conditions coupled to electrospray tandem mass spectrometry

A sensitive and specific LC-MS/MS assay for the quantitative determination of EO9 and its metabolite EO5a is presented. A 200-microl human plasma aliquot was spiked with a mixture of deuterated internal standards EO9-d3 and EO5a-d4 and extracted with 1.25 ml ethyl acetate. Dried extracts were reconstituted in 0.1 M ammonium acetate-methanol (7 : 3, v/v) and 25 microl-volumes were injected into the HPLC system. Separation was achieved on a 150 x 2.1 mm C18 column using an alkaline eluent (1 mM ammonium hydroxide-methanol (gradient system)). Detection was performed by positive ion electrospray followed by tandem mass spectrometry. The assay quantifies a range from 5 to 2500 ng/ml for EO9 and from 10 to 2500 ng/ml EO5a using 200 microl of human plasma samples. Validation results demonstrate that EO9 and EO5a concentrations can be accurately and precisely quantified in human plasma. This assay will be used to support clinical pharmacologic studies with EO9.     

A Simple and Rapid Reversed Phase High Performance Liquid Chromatographic Method for Quantification of Alprazolam and α-Hydroxyalprazolam in Plasma

Abstract

A simple and rapid reversed-phase liquid chromatographic method for the determination of alprazolam and a-hydroxyalprazolam in plasma is described. Flunictrazepam was used as internal standard. Plasma samples were buffered with sodium borate and extracted with dichloromethane /n-pentane 4:6 v/v for 60 sec on a vortex apparatus. Extraction solvent was evaporated to dryness and extraction residues were reconstituted in the mobile phase. Samples were chromatographed on a 5μ Lichrospher RP-18 column (25cm × 4mm i. d) using acetonitrile/water 40:60 v/v as the mobile phase. The column effluent was monitored at 230nm. The lower limit of detection was 1ng/ml for alprazolam and a-hydroxyalprazolam while the lower limit of quantification was 2ng/ml for both compounds. Peak height and plasma     

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.     

Determination of a potential anxiolytic drug (CGS 20625) in human plasma by high-performance liquid chromatography.

An analytical method employing reversed-phase high-performance liquid chromatography is described for the determination of a potential anxiolytic agent in human plasma. This experimental drug candidate has potent and selective affinity for the central benzodiazepine receptor complex. The compound and internal standard are extracted from buffered plasma (pH 9.0) into ethyl acetate. The solvent is evaporated and the residue is reconstituted in chromatographic mobile phase. Separation is achieved on a 5-microns phenyl column with ultraviolet absorbance detection of the drug and internal standard at 270 nm. Recovery and reproducibility assessments indicate good accuracy (overall relative recovery of 101%) and precision (coefficients of variation from 2.0 to 11%) over the concentration range 10-1000 ng/ml. The limit of quantification for the method is 10 ng/ml. The method is suitable for pharmacokinetic analysis following the administration of 80 mg of drug to normal volunteers.     

Simultaneous determination of vinburnine and 6-hydroxyvinburnine in human plasma by high-performance liquid chromatography

An isocratic high-performance liquid chromatographic method has been developed to allow the simple and rapid determination of both vinburnine (I) and its main metabolite, 6-hydroxyvinburnine (II), in heparinized human plasma (0.5 ml). Compounds I and II and p-chlorodisopyramide (internal standard) were first extracted with alkalinized ethyl acetate and then with sulphuric acid. Separation was achieved on a reversed-phase muBondapak C18 column with a mobile phase of acetonitrile-water-0.1 M heptanesulphonate in acetic acid and with detection at 254 nm. Each run required 20 min. The within-day coefficients of variation for identical samples (20 ng/ml) were 7 and 6% and between-day coefficients of variation 8 and 26% for I and II, respectively. The detection limit was 5 ng/ml (normal therapeutic concentration, 10-300 ng/ml). The application of the method to drug monitoring was compared to that of a thin-layer chromatographic procedure.     

Simple high-performance liquid chromatographic method for the determination of medroxyprogesterone acetate in human plasma

The high-performance liquid chromatographic (HPLC) method was developed as a simple, reliable alternative to available methods for measuring plasma concentrations of medroxyprogesterone acetate (MPA). The HPLC method has been successfully automated and is suitable for the rapid, inexpensive analysis of large batches of plasma samples. The best approach involves a solvent extraction followed by HPLC separation and analysis. MPA can be efficiently extracted, at all pH values, by nonpolar solvents. The Spherisorb 5-ODS2 HPLC column provides excellent separation of MPA from endogenous steroids of similar structure and from extraneous plasma blank peaks. A batch of 30-40 samples can be prepared by HPLC analysis in 2-3 hours, with a chromatographic run time of 10 minutes/sample. Calibration curves between 5-250 ng/ml show a good correlation between peak height ratio and MPA concentration, even at low levels. Plasma concentrations of MPA in patients receiving 1 g/day were between 12.6-270 ng/ml in this study, suggesting that the sensitivity of this method, 10 ng/ml, is sufficient for monitoring therapeutic concentrations of MPA. The results show a wide individual variation in plasma concentrations following similar dosing schedules--a finding reported by other workers.     

High-performance liquid chromatographic determination of naltrexone in plasma of hemodialysis patients

A simple, sensitive, selective and reliable reversed-phase high-performance liquid chromatographic (HPLC) method with UV detection is described for the determination of naltrexone in plasma samples. Naltrexone and the internal standard, naloxone, were isolated from plasma either with a liquid-liquid extraction method using ethyl acetate or with a solid-phase extraction method using Sep-Pack C18 cartridge before chromatography. The extracts were dried under a stream of nitrogen and the samples were reconstituted in the mobile phase, then 20 microL were injected on a Waters Symmetry C18 column (5 microm particle size, 4.6 x 150 mm). The mobile phase consisted of 0.06% triethylamine (pH 2.8)-acetonitrile (92:8, v/v) pumped at 1 mL/min. The peak-area ratio versus plasma concentration was linear over the range of 10-500 ng/mL and the detection limit was less than 8 ng/mL. Quantification was by ultra-violet detection at 204 nm. The present method was applied to the determination of the plasma concentration of naltrexone in dialyzed patients. Patients (n = 8) with severe generalized pruritus received 50 mg of naltrexone orally per day for 2 weeks. The variability in the therapeutic response in treated patients required plasma concentration investigations of this opioid antagonist.     

Determination of nadolol in serum by high-performance liquid chromatography with fluorimetric detection.

A sensitive high-performance liquid chromatographic method for a routine assay of nadolol in serum is described. Serum samples spiked with atenolol (internal standard) were extracted with diethyl ether. After centrifugation, the organic layer was evaporated to dryness. The residue was redissolved in the mobile phase and injected onto an octadecyl silica column (150 mm x 4.6 mm I.D.). The mobile phase was 0.05 M ammonium acetate (pH 4.5)-acetonitrile (85:15, v/v). Fluorometric detection (excitation 230 nm, emission 300 nm) was used. The minimum detectable level of nadolol in serum was 1 ng/ml.     

High-performance liquid chromatographic method for routine determination of vitamins A and E and beta-carotene in plasma.

A simple and reliable reversed-phase high-performance liquid chromatographic (HPLC) method for the routine determination of vitamins A and E and beta-carotene in plasma (or serum) with wavelength-programmed ultraviolet-visible absorbance detection is described. A 200-microliters aliquot of serum or plasma sample, after deproteinization with ethanol, and containing tocopherol acetate as internal standard, was extracted with butanol-ethyl acetate. Sodium sulphate was added for dehydration. Analytes of extracted samples were found to be stable for at least four days. A 10-microliters aliquot of this organic extract was used for HPLC analysis. The mobile phase was methanol-butanol-water (89.5:5:5.5, v/v) and the flow-rate was set at 1.5 ml/min. The analytes of interest were well separated from other plasma constituents within 22 min at 45 degrees C. The lowest detection limits of vitamins A and E and beta-carotene were 0.02, 0.5 and 0.1 microgram/ml, respectively. The recovery and reproducibility of the present method were around 90%. The method is sensitive, specific and can be used for epidemiological studies and for routine determination of vitamin deficiency. Several important factors that may affect the analysis are also discussed in this paper.     

Determination of cetirizine in human urine by high-performance liquid chromatography.

A high-performance liquid chromatographic method for the determination of the histamine H1-receptor antagonist cetirizine in human urine was developed. Cetirizine and the internal standard are extracted from acidified (pH 5) urine (0.5 ml) into chloroform and the organic layer is evaporated to dryness. The residue is chromatographed on a Spherisorb 5ODS-2 column using Pic A (5 mM aqueous tetrabutylammonium phosphate)-methanol-tetrahydrofuran (33:65:2, v/v) as the mobile phase with ultraviolet detection (230 nm). The calibration graph is linear from 0.1 to 10 micrograms/ml and using 0.5 ml of urine the detection limit is 20 ng/ml. The within-run relative standard deviation is less than 6% and the accuracy is within 10% of the theoretical value at concentrations between 0.1 and 10 micrograms/ml in urine. There is a good correlation (r = 0.99606) with a previously described capillary gas chromatographic assay.     

Determination of glycyrrhetinic acid in human plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method has been developed for measuring 18 beta-glycyrrhetinic acid (GRA) in human plasma in the range of 0.1-3 micrograms/ml. The acetate ester of GRA is added to the plasma as an internal standard, plasma proteins are denatured with urea to release GRA, and the GRA and the internal standard are extracted in an ion-pairing solid-phase extraction process. An isocratic, reversed-phase HPLC separation is used, followed by ultraviolet absorbance detection at 248 nm. The results from the analysis of five GRA-fortified plasma pools show a mean relative standard deviation of 7% and are accurate to within 10%. With evaporative concentration of the extract, the limit of detection for GRA in plasma is approximately 10 ng/ml.     

Quantitative determination of norethisterone acetate in human plasma by capillary gas chromatography with mass-selective detection

An analytical method for the determination of norethisterone acetate (NETA) in human plasma by capillary gas chromatography-mass-selective detection (GC-MS), with testosterone acetate as internal standard, was developed and validated. After addition of the internal standard, the compounds were extracted from plasma at basic pH into diethyl ether-dichloromethane (3:2 v/v), which was then evaporated to dryness. The compounds were converted into their pentafluoropropionyl derivatives which were determined by gas chromatography using a mass selective detector at m/z 486 for NETA and m/z 476 for the internal standard. Intra-day and inter-day accuracy and precision were found suitable over the range of concentrations between 0.10 to 10 ng/ml. The method was applied to clinical samples.