Isocratic Reversed-phase High-performance Liquid Chromatography Research Articles

Determination of paclitaxel in human plasma using single solvent extraction prior to isocratic reversed-phase high-performance liquid chromatography with ultraviolet detection

An isocratic reversed-phase high-performance liquid chromatographic method with ultraviolet detection at 230 nm has been developed for the determination of paclitaxel in human plasma. Plasma samples were prepared by a selective one-step liquid–liquid extraction involving a mixture of acetonitrile– n-butyl chloride (1:4, v/v). Paclitaxel and the internal standard docetaxel were separated using a column packed with ODS-80A material, and a mobile phase consisting of water–methanol–tetrahydrofuran–ammonium hydroxide (37.5:60:2.5:0.1, v/v). The calibration graph for paclitaxel was linear in the range 10–500 ng/ml, with a lower limit of quantitation of 10 ng/ml, using 1 ml plasma samples. The extraction recoveries of spiked paclitaxel and docetaxel to drug-free human plasma were 89.6±8.52 and 93.7±5.0%, respectively. Validation data showed that the assay for paclitaxel is sensitive, selective, accurate and reproducible. The assay has been used in a single pharmacokinetic experiment in a patient to investigate the applicability of the method in vivo.

Assessing the Fate of Organic Contaminants in Aquatic Environments: Mechanism and Kinetics of Hydrolysis of a Carboxylic Ester

An integrated experiment is proposed for an upper level under-graduate environmental chemistry laboratory. Using 2,4-dinitrophenylacetate (DNPA) as an example, the mechanisms of the abiotic hydrolysis of carboxylic esters are presented and the kinetic expressions are derived. The proposed experimental set-up uses isocratic reversed phase HPLC with UV detection to follow the decrease of DNPA concentration during hydrolysis experiments. The Arrhenius parameters of the rate constants for the neutral and base-catalyzed abiotic hydrolysis are evaluated using hydrolysis rates obtained at various pH values and temperatures. With these parameters, the half-life of the compound under given environmental conditions can be assessed. The experiment is very well suited to introduce or repeat important basic concepts of kinetics and instrumental analysis and to sensitize the students for the often complex nature of environmental processes.

Determination of naringin and naringenin in human urine by high-performance liquid chromatography utilizing solid-phase extraction

An HPLC method for determining a flavonoid naringin and its metabolite, naringenin, in human urine is presented for application to the pharmacokinetic study of naringin. Isocratic reversed-phase HPLC was employed for the quantitative analysis by using hesperidin for naringin or hesperetin for naringenin as internal standard and solid-phase extraction using a strong anion exchanger, Sep-Pak Accell QMA cartridge. The HPLC assay was carried out using an Inertsil ODS-2 column (250×4.6 mm I.D., 5 μm particle size). The mobile phases were acetonitrile–0.1 M ammonium acetate–acetic acid (18:81:1, v/v; pH 4.7) for naringin and acetonitrile–0.1 M ammonium acetate–triethylamine (25:75:0.05; v/v; pH 8.0) for naringenin. The flow-rate was 1.0 ml min −1. The analyses were performed by monitoring the wavelength of maximum UV absorbance at 282 nm for naringin and at 324 nm for naringenin. The lower limits of quantification were ca. 25 ng/ml for naringin and naringenin with R.S.D. less than 10%. The lower limits of detection (defined as a signal-to-noise ratio of about 3) were approximately 5 ng for naringin and 1 ng for naringenin. A preliminary experiment to investigate the urinary excretion of naringin, naringenin and naringenin glucuronides after oral administration of 500 mg of naringin to a healthy volunteer demonstrated that the present method was suitable for determining naringin and naringenin in human urine.

Analysis of recombinant human growth hormone directly in osmotic shock fluids

An isocratic reversed-phase high-performance liquid chromatography (RP-HPLC) method for the determination of human growth hormone (hGH) directly in osmotic shock fluids is described. This methodology allows an initial rapid evaluation of the quality and quantity of hGH being secreted in the bacterial periplasmic space right after, or even during fermentation. Considering that RP-HPLC does not identify size isomers, these were determined via a parallel run of the same osmotic shock fluid on high-performance size-exclusion chromatography, coupled with radioimmunoassay, of the eluted fractions. The methodology provides a complete picture, within 24 h from the beginning of the fermentation process, of the recombinant protein being produced with respect to its activity, identity, yield, and hGH-related contaminants. These latter include sulfoxide and desamido derivatives, dimer and high-molecular-mass forms.

An HPLC/RIA method for dynorphin A1-13 and its main metabolites in human blood

A selective HPLC/RIA procedure for the determination of dynorphin A1-13 (Dyn A1-13) and its major metabolites in human blood was developed. In order to block peptidase activity, blood samples were transferred into an aliquot of a blocking solution (5% aqueous ZnSO 4 solution-acetonitrile-methanol; 5:3:2, v/v/v). After solid phase extraction, reconstituted aliquots were injected into an isocratic reversed phase HPLC system to separate Dyn A1-13 from its main metabolites (Dyn A2-13, Dyn A1-12 and Dyn A2-12). The isolated and concentrated HPLC-fractions were assayed by RIA using a commercially available antiserum. Intra-day variabilities for quality controls (0.07, 0.25, and 1 ng ml −1) of Dyn A1-13, A2-13, A1-12, A2-12 were between 9 and 41%. Accuracy was between 86 and 132%. Inter-day variability for single quality controls analyzed on five days for Dyn A1-13, A2-13, A1-12, A2-12 was between 4 and 49% for 0.07, 0.25 and 1 ng ml −1 samples, respectively. Accuracy was between 72 and 129%. Five different batches of control blood showed blood levels no different from zero. Considering the complexity of the assay, the method is selective, accurate and reproducible with a limit of detection of 0.07 ng ml −1 for Dyn A1-13, Dyn A2-13, Dyn A1-12 and 0.21 ng ml −1 for Dyn A2-12. The assay was applied to the determination of Dyn A1-13 and its metabolites in blood samples of 2 subjects receiving i.v. infusions of 250 μg or 1000 μg kg −1 Dyn A1-13 over 10 min.

High-performance liquid chromatographic determination of 4-methylpyrazole in plasma and in dialysate

A rapid method for the determination of 4-methylpyrazole (4-MP) levels in plasma and in dialysate by isocratic reversed-phase high-performance liquid chromatography with UV detection is described. The internal standard was the 3-methylpyrazole (3-MP). Plasma sample preparation consisted of a protein precipitation. Dialysate samples were injected without preparation. The method was linear up to 30 mg l −1 in plasma and up to 5 mg l −1 in dialysate. The within-day precisions (C.V.) were less than 4% in plasma and were less than 2% in dialysate. The day-to-day precisions (C.V.) were less than 7% in plasma and were less than 3% in dialysate. This method is easy to perform and has practical interest for clinicians who need to monitor in emergency 4-MP levels in ethylene glycol and methanol poisonings.

Efficient high-performance liquid chromatographic assay for the simultaneous determination of metoprolol and two main metabolites in human urine by solid-phase extraction and fluorescence detection

An improved, more efficient method for the determination of metoprolol and its two metabolites in human urine is reported. The simultaneous analysis of the zwitterionic metoprolol acidic metabolite ( III, H117/04) with the basic metabolites α-hydroxymetoprolol ( II, H119/66), metoprolol ( I) and guanoxan ( IV, internal standard) was achieved employing solid-phase extraction and isocratic reversed-phase HPLC. The analytes were extracted from urine (100 μl) using C 18 solid-phase extraction cartridges (100 mg), and eluted with aqueous acetic acid (0.1%, v/v)–methanol mixture (40:60, v/v, 1.2 ml). The eluents were concentrated (250 μl) under vacuum, and aliquots (100 μl) were analysed by HPLC with fluorescence detection at 229 nm (excitation) and 309 nm (emission) using simple isocratic reversed-phase HPLC (Novapak C 18 radial compression cartridge, 4 μm, 100×5 mm I.D.). Acetonitrile–methanol–TEA/phosphate buffer pH 3.0 (9:1:90, v/v) was employed as the eluent (1.4 ml/min). All components were fully resolved within 18 min, and the calibration curves for the individual analytes were linear ( r 2≥0.996) within the concentration range of 0.25–40.0 mg/ml. Recoveries for all four analytes were greater than 76% ( n=4). The assay method was validated with intra-day and inter-day variations less than 2.5%.

Urinary excretion of the TRH-like peptide pyroglutamyl-glutamyl-prolineamide in rats.

TRH-like immunoreactivity (TRH-LI) was estimated in methanolic extracts of rat tissues and blood by RIA using antiserum 4319, which binds most peptides with the structure pGlu-X-ProNH2, or antiserum 8880, which is specific for TRH (pGlu-His-ProNH2). TRH-LI (determined with antiserum 4319) and TRH (determined with antiserum 8880) contents were 8 and 8 ng/g in brain, 216 and 222 ng/g in hypothalamus, 6.5 and 6 ng/g in pancreas, 163 and 116 ng/g in male pituitary, 105 and 77 ng/g in female pituitary, 1 and 0.1 ng/g in salivary gland, 61 and 42 ng/g in thyroid, 12 and 3 ng/g in adrenal, 3 and 0.3 ng/g in prostate, and 11 and 0.8 ng/g in ovary respectively. Blood TRH-LI (antiserum 4319) and TRH (antiserum 8880) levels were 31 and 18 pg/ml in male rats, and 23 and 10 pg/ml in female rats respectively. Unextracted serum obtained from blood kept for at least 1 h at room temperature no longer contained authentic TRH but still contained TRH-LI (males 20.3 +/- 3.1, females 15.9 +/- 3.0 pg/ml; means +/- S.E.M.). Isocratic reverse-phase HPLC showed that TRH-LI in serum is largely pGlu-Glu-ProNH2 (< EEP-NH2), a peptide previously found in prostate and anterior pituitary. In urine, TRH-LI (antiserum 4319) and TRH (antiserum 8880) levels were 3.21 +/- 0.35 and 0.32 +/- 0.04 ng/ml in male rats and 3.75 +/- 0.22 and 0.37 +/- 0.04 ng/ml in female rats respectively (means +/- S.E.M.). Anion-exchange chromatography on QAE-Sephadex showed that urine of normally fed rats contains both basic/neutral TRH-LI (b/n TRH-LI) and acidic TRH-LI (aTRH-LI) in a ratio of approximately 40:60, and further analysis by HPLC indicated that aTRH-LI represents < EEP-NH2. Analysis of food extracts and urine from fasted rats demonstrated that b/n TRH-LI is derived from food particles spilled by the rats during urine collection, while aTRH-LI is endogenously produced. While urinary aTRH-LI levels were higher in female than in male rats (2.99 +/- 0.41 vs 2.04 +/- 0.20 ng/ml), the daily urinary excretion was similar in both sexes (females 15.6 +/- 1.4, males 19.5 +/- 2.0 ng/day). Intravenously injected < EEP-NH2 disappeared from serum with a half-life of approximately 1 h, and was recovered unchanged and quantitatively in urine. In contrast, when < EEP-NH2 was administered with food, only approximately 0.5% was recovered in urine. The urinary clearance rate of serum TRH-LI amounted to 0.52 +/- 0.10 ml/min in males and 0.34 +/- 0.05 ml/min in females. In view of the presence of < EEP-NH2 in the anterior pituitary gland, and the regulation of its content in parallel with gonadotrophins, we examined the possibility that serum < EEP-NH2 is of pituitary origin and correlates with gonadotrophin secretion. However, treatments that alter pituitary < EEP-NH2 content and gonadotrophin release had no effect on serum TRH-LI or urinary aTRH-LI. In conclusion, the TRH-like peptide < EEP-NH2 is present in rat serum and is excreted into the urine. Moreover, < EEP-NH2 in serum and urine is not derived from rat food and is probably not of pituitary origin.

Simultaneous determination of low plasma concentrations of retinol and tocopherols in preterm infants by a high-performance liquid chromatographic micromethod

A method for the simultaneous determination of low concentrations of retinol and tocopherols from 100 μl plasma using isocratic reversed-phase HPLC is described. Retinol is quantified with a programmable UV-Vis detector, whereas tocopherols are quantified by fluorescence detection using tocol as the internal standard. Intra- and inter-assay precision are 3.7 and 4.3% for retinol and 2.3 and 6.1% for α-tocopherol, respectively. The accuracy as determined with standard material from the US National Institute of Standards and Technology with low, medium and high concentrations is in the range of 0.2–6.0% bias for retinol and of −3.0 to 5.5% for α-tocopherol, respectively. This method is highly sensitive and selective and has a good precision and accuracy for measuring low concentrations of vitamins in small plasma volumes.

High performance liquid chromatographic behaviour of several barbiturates on a newly developed high purity silica column

A simple isocratic reversed phase HPLC method for the separation of a series of barbiturates is described. The column used is a newly developed synthetic silica known as Symmetry Clg (Waters Corp., Milford, MA, USA) and the mobile phase consists of 50 mM KH2PO4 (pH 4.55): acetonitrile water (20:30:50:). The barbiturates studied are monitored by UV detection at 225 nm. Detailed methodology and the advantage of the Symmetry C18 reversed phase is presented.

GlutathioneS-Transferase-Catalyzed Conjugation of Bioactivated Aflatoxin B1in Rabbit Lung and Liver

Aflatoxin B1(AFB1) requires bioactivation to AFB1-8,9-epoxide for carcinogenicity, and glutathioneS-transferase (GST)-catalyzed conjugation of activated AFB1with glutathione (GSH) is a critical determinant of susceptibility to the mycotoxin. Incubations containing [3H]AFB1, rabbit liver microsomes, an NADPH-generating system, 1 mMGSH, and GST-containing lung or liver cytosol were performed to assess the abilities of lung and liver GSTs to conjugate AFB1-8,9-epoxide. [3H]AFB1–GSH was isolated by isocratic reverse-phase high-performance liquid chromatography (HPLC) and quantitated by liquid scintillation spectroscopy. Maximal [3H]AFB1–GSH formation rates were significantly lower for lung than for liver (0.3 ± 0.1 and 1.7 ± 0.4 nmol/mg/hr, respectively). Immunoprecipitation of rabbit pulmonary cytosolic GSTs with anti-alpha or anti-mu GST antisera decreased [3H]AFB1–GSH production by approximately 45 and 51%, respectively, indicating that alpha-class and mu-class GSTs are of similar importance in catalyzing this reaction in the lung. Because mu-class GSTs comprise only a small proportion of total lung GST content, these enzymes have high specific activity toward AFB1-8,9-epoxide. In contrast, the pi-class GST appeared to play a negligible role. Using a rat liver microsomal system to generate both AFB1exo- andendo-epoxide isomers, and analysis based on chiral HPLC, we found that rabbit liver cytosolic GSTs catalyzed formation of both AFB1exo- andendo-epoxide–GSH conjugates, whereas pulmonary cytosolic GSTs catalyzed formation of only theexostereoisomer at detectable levels. Despite a preference for conjugating the more mutagenic AFB1exo-epoxide isomer, the relatively low capacity for GST-catalyzed detoxification of bioactivated AFB1in lung may be an important factor in the susceptibility of the lung to AFB1toxicity.

High-performance liquid chromatographic determination of cyclohexylhydantoin and cyclohexylidenehydantoin formed during the synthesis of phenylhydantoin from hydantoin and cyclohexanone

dl-Phenylhydantoin can be converted into d-phenylglycine by an enzymatic process. d-phenylglycine is an important starting material in the production of β-lactams such as semisynthetic penicillins and cephalosporins. In our laboratory, the synthesis of phenylhydantoin was achieved from hydantoin and cyclohexanone in the presence of a base. An efficient and fast isocratic reversed-phase high-performance liquid chromatography method was developed for the determination of phenylhydantoin, cyclohexylhydantoin and cyclohexylidenehydantoin. Quantitative analysis was carried out by an external standard method.

Determination of naringin and naringenin in human plasma by high-performance liquid chromatography.

An HPLC method for determining a flavonoid, naringin, and its metabolite, naringenin, in human plasma is presented for application to the pharmacokinetic study of naringin. Isocratic reversed-phase HPLC was employed for the quantitative analysis by using genistin (for naringin) or diadzein (for naringenin) as an internal standard and solid-phase extraction using s Sep-Pak t C18 cartridge. For the determination, HPLC was carried out using an Inertsil ODS-2 column (250 x 4.6 m I.D., 5 microns particle size). The mobile phases were acetonitrile-0.1 M ammonium acetate solution (20:80, v/v; pH 7.1) for naringin and acetonitrile-0.1 M ammonium acetate solution-acetic acid (30:69:1, v/v; pH 4.9) for naringenin. The flow-rate was 1 ml min-1. The analyses were performed by monitoring the wavelength of maximum UV absorbance at 280 nm for naringin and at 292 nm for naringenin. The detection limits on-column were about 0.2 ng for the two flavonoids.

Determination of capsaicinoids in low pungency paprika

Capsaicin, dihydrocapsaicin and nordihydrocapsaicin were determined in paprika powders and oleoresins after clean-up on silica and aluminium oxide with controlled activity followed by isocratic reverse-phase HPLC (RP-HPLC) with fluorescence detection. Caprylic acid vanillylamide (CVA) was synthesized for use as internal standard. Samples with less than 0.6 μg/g (10 Scoville Heat Units) were analysed.

Determination of sumatriptan succinate in human plasma by high-performance liquid chromatography with coulometric detection and utilization of solid-phase extraction

Sumatriptan succinate (the analyte) and naloxone (the internal standard) were extracted from plasma with a solid-phase extraction technique. Chromatography and detection were performed by isocratic reversed-phase high-performance liquid chromatography with coulometric end-point detection. The standard curve was linear over the range 0–100 ng/ml of sumatriptan succinate in plasma. The reproducibility (as defined by the coefficient of variation, C.V.) over the range of the standard curve was 4.9–7.3%. The recovery averaged 83%. The sensitivity was 0.25 ng of sumatriptan on column (allowing a concentration of 0.5 ng/ml to be determined from a 1-ml plasma sample volume). Plasma profiles of the analyte following subcutaneous (s.c.) administration in eight normal male volunteers, are presented.

P-5-22 - Effects of selective antidepressants on the sensitivity of central adrenergic receptors

Therapeutic drug monitoring necessitates efficient, fast and reliable analytical methods validated by external quality control. We therefore devised an isocratic reversed-phase HPLC method with ultraviolet detection and optimised this to quantify mirtazapine, reboxetine, moclobemide, venlafaxine, O-desmethylvenlafaxine, paroxetine, fluvoxamine, fluoxetine, norfluoxetine, sertraline, citalopram, amitriptyline, nortriptyline, imipramine, desipramine, doxepin, nordoxepin, clomipramine, norclomipramine, trimipramine, mianserine, maprotiline, normaprotiline, amisulpride, clozapine, norclozapine, quetiapine, risperidone and 9-OH-risperidone in human serum. After solid-phase extraction of the drugs and metabolites, the chromatographic separation was achieved on a Nucleosil 100-Protect 1 column with acetonitrile–potassium dihydrogenphosphate buffer as mobile phase. The method was validated for therapeutic and toxic serum ranges. A linear relationship (r>0.998) was obtained between the concentration and the detector signal. Recoveries were between 75 and 99% for the drugs and metabolites. The accuracy of the quality control samples, expressed as percent recovery, ranged from 91 to 118%; intra- and inter-assay-relative standard deviations were 0.9–10.2% and 0.9–9.7%, respectively. Additional external quality control is carried out since 3 years. This method is applicable to rapidly and effectively analyze serum or plasma samples for therapeutic drug monitoring of about 30 antidepressants and atypical antipsychotics.

Determination of naloxone in human plasma by high-performance liquid chromatography with coulometric detection

Naloxone, the analyte and the internal standard, sumatriptan, are extracted from plasma using solid-phase extraction columns. Chromatography and detection are performed using isocratic reversed-phase high-performance liquid chromatography (HPLC) with coulometric end-point detection. The standard curve was linear over the range 0–50 ng/ml of naloxone in plasma. The reproducibility, the coefficient of variation (C.V.) of the method over the range of the standard curve was 6.2–11.2%. The recovery averaged 90.4±8.9%. A plasma profile following i.v. administration of naloxone in one normal healthy volunteer is presented.

Kinetic determination of morphine in illicit powders, using a fluoride selective electrode, based on reaction with 1-fluoro-2,4-dinitrobenzene

A simple, accurate and selective method is described for the determination of morphine in illicit powders, based on monitoring the initial rate of fluoride ion liberation from the reaction of morphine with 1-fluoro-2,4-dinitrobenzene at pH 9 and 35 °C, using a solid-state fluoride ion-selective electrode. Under optimized reaction conditions, as little as 15 μg/ml of morphine is determined with an average recovery of 99.5% and a mean relative standard deviation of 1.2%. Determination of morphine in real illicit powders containing 6–18% morphine gives results comparing favorably with those obtained from isocratic reverse-phase high-performance liquid chromatography. No interferences are caused by many structurally related and associated compounds such as codeine, acetylcodeine, ethylmorphine, acetylmorphine and diacetylmorphine (heroin).

Determination of Tranylcypromine in Urine and Pharmaceutical Formulation by HPLC Using Symmetry column

Abstract A simple isocratic reversed phase HPLC method for the determination of tranylcypromine sulfate (TCP) is described. The column used is Symmetry C18 (Waters Corp., Milford, MA, USA) and the mobile phase consists of 50 mM KH2PO4 (pH 4.55):methanol:water (20:10:70). Tranylcypromine sulfate is monitored by U.V. detection at 264 nm. The calibration curve of tranylcypromine sulfate is constructed over the range 25 nmol – 375 nmol/mL with a correlation coefficient 0.999. The method is specific and sensitive with a lower limit of detection 5 nmol/mL, the intra-assay and inter-assay relative standard deviation were below 10%. The validated assay procedure was applied to determine the tranylcypromine sulfate in urine and also to evaluate TCP in pharmaceutical preparation. Detailed methodology and the advantage of this C18 reversed phase is presented.

Simultaneous quantitation of buspirone and its major metabolite 1-(2-pyrimidinyl)piperazine in human plasma by high-performance liquid chromatography with coulometric detection

Buspirone is a member of the azapirone group of anxiolytic drugs and has one major metabolite, 1-(2-pyrimidinyl)piperazine (1-PP). The analyte, its metabolite and the internal standard were extracted from plasma utilizing solid-phase extraction columns. Chromatography was performed using isocratic reversed-phase high-performance liquid chromatography with coulometric end-point detection. The calibration graph was linear over the range 0–50 ng ml −1 of plasma. The lower limits of quantitation for buspirone and 1-PP were 0.5 and 2 ng ml −1, respectively, when 1 ml of plasma was extracted. The intra-assay relative standard deviations (RSD) over the range of the calibration graph varied from 4 to 12.5% for buspirone and 1-PP. The inter-assay RSD was 6.9% for 1-PP and 9.6% for buspirone. The recovery averaged 96% for buspirone and 66% for 1-PP. Plasma profiles of buspirone and 1-PP following oral dosing are presented.