μBondapak Phenyl Column Research Articles

Developing a method for the quantitative determination of the P-glycoprotein activity marker fexofenadine in blood plasma

A highly sensitive and well-reproducible HPLC procedure has been developed for the quantitative determination of fexofenadine in the blood plasma. The drug was separated by reverse-phase HPLC on a μ-Bondapak Phenyl column and reliably detected by spectrophotometry at 220 nm.

High-performance liquid chromatographic assay with fluorescence detection for the routine monitoring of the antidepressant mirtazapine and its demethyl metabolite in human plasma

A validated HPLC method for the simultaneous quantitative analysis of the antidepressant mirtazapine and its demethyl metabolite in human plasma is described. The active constituents including internal standard were extracted from 1 ml of plasma with hexane and separated on a μBondapak Phenyl column with fluorescence detection. The lower limit of quantification was 0.5 ng/ml, without significant interferences with endogenous or exogenous components. Inter- and intra-assay accuracy determined at quality control levels of 2, 10 and 80 ng/ml were, respectively, 104.6–113.7% and 105.1–117.7% for mirtazapine, and 91.7–99.3% and 89.9–103.7% for demethylmirtazapine. In all cases the precision was below 6.8%.

On the isolation and characterisation of a crustacean hyperglycaemic hormone in the eyestalk of the shrimp Penaeus indicus

In decapod crustaceans, the eyestalk represents the pivotal source for several neuropeptides influencing various physiological activities concerned with metabolism, growth and reproduction. Prominent among them is the crustacean hyperglycaemic hormone (CHH) which regulates the blood glucose level by its `diabetogenic' action. This paper reports on the first characterisation of a CHH in the commercially important penaeid species Penaeus indicus. Reversed phase HPLC of extracts from eyestalk ganglia on a Waters μ-Bondapak phenyl column combined with an enzyme linked immunosorbent assay revealed that among six immunoreactive peptide fractions in total a late eluting peak fraction with the retention time of about 50 min shows the strongest immunoreactivity to a polyclonal rabbit antiserum raised against the CHH of the freshwater crayfish Orconectes limosus. The amino acid composition of this peptide, purified by rechromatography was determined according to the orthophthaldialdehyde post column derivatisation method. The most interesting features are the low molar concentration of acidic amino acid residues (e.g. Asx, Glx) but a high molar concentration of lysine residues. The CHH material was localised immunocytochemically in the neurosecretory centres of X-organ sinus gland system using the same antiserum. A large number of perikarya of the medulla terminalis X-organ, the neural tract as well as the sinus gland showed intense immunoreactivity. Though the hyperglycaemic response of this HPLC purified CHH of P. indicus was found to be very poor when injected into eyestalkless as well as eyestalk-intact crayfish, O. limosus, it clearly induced hyperglycaemia in P. indicus.

High Performance Liquid Chromatographic Analysis of The Cardioprotective Agent Dexrazoxane in Human Plasma and Urine

For the purpose of a pharmacokinetic study in the comparison of two intravenous pharmaceutical formulations of the cardioprotective agent dexrazoxane, we have developed an High Performance Liquid Chromatographic (HPLC) assay to quantify the drug in human plasma and urine. The plasma sample pretreatment involved a protein precipitation step with acetonitrile followed by an extraction with 10% 2-methyl-2-propanol in chloroform (v/v). Urine samples were diluted in distilled water and subsequently extracted with 10% 2-methyl-2-propanol in chloroform (v/v). After evaporation of the organic solvents, the residues were dissolved and analysed on a μBondapak Phenyl column with a mobile phase consisting of 0.01 M potassium phosphate pH 4.7 and methanol (8:2, v/v). Detection was performed at 208 nm. The lower limit of quantitation was 0.1 μg/mL and 10 μg/mL for plasma and urine respectively, using 1.0 mL sample volumes. The usefulness of the method has been demonstrated in clinical samples originating from patients treated with dexrazoxane. Dexrazoxane is not stable in plasma at ambient temperature: after 6.5 hours the initial concentration was 42.6 ± 1.0% (n=3) of the original concentration of 100 μg/mL. After sampling in the clinic, plasma samples should be stored immediately at −30°C. Under these conditions dexrazoxane is stable for at least 5 months. In urine the drug is stable for 24 hours when stored at 4–8°C. An aliquot of the voided urine sample can be stored at -30°C for at least 4 months without drug decomposition.

Liquid Chromatographic Separation and Indirect Detection of Azacrown Compounds Using α-Naphthalene Sulfonic Acid as a Detection Reagent

Indirect detection and high-performance liquid chromatographic separation of azacrown compounds, such as 1-aza-15-crown-5 and 1,7-dioxa-4,10,13-triazacyclopentadecane, were studied. The indirect detection reagent used was α-naphthalene sulfonic acid. The elution behaviors of the azacrown compounds on a μ-Bondapak phenyl column with varying pH and different methanol and α-NSA concentrations were also elucidated.

A neuropeptide family from the sinus gland of the Mexican crayfish, Procambarus bouvieri (Ortmann)

The main neuroendocrine axis of crustaceans is constituted by the brain, medulla terminalis X-organ (MTXO) -sinus gland (SG) system. A variety of hormonal peptides are synthesized in the MTXO where the peptidergic neuronal somata are situated. The peptides are then transported via axonic flow to the SG, which is a conglomerate of bulbous axonic terminals in close contact with the hemolymph, where these hormones are liberated to the circulation by a process of exocytosis. Four peptides have been isolated from crude aqueous extracts of microdissected SG of the Mexican crayfish, Procambarus bouvieri, by means of a single-step reverse-phase high pressure liquid chromatography (RP-HPLC) on a μBondapak-Phenyl column; these have been characterized physiologically by specific bioassays, and structurally by means of tryptic peptide mapping and sequencing. These procedures have identified (in the order of elution) a gonad-inhibiting hormone (GIH), a molt-inhibiting hormone (MIH), a major isomorph of the crustacean hyperglycaemic hormone (CHH-I) and its minor isomorph (CHH-II), in approximate concentrations of 5, 18, 60, and 20 ng per SG, respectively. Their common characteristics are: (1) acidic pl, (2) hydrophobicity, (3) molecular masses between 8300 and 8400 Da, (4) approximately 72 amino acid residues, (5) blocked N- and C-termini, (6) six cysteines forming three disulfide bridges, (7) lack of histidine, methionine, and tryptophan residues, (8) long tracts of sequence identity. These features clearly establish these four peptides as members of one neuropeptide family. Recently, a factor with gonad-stimulating activity has been isolated from this same crude extract by means of RP-HPLC. This factor stimulated by 300% the specific incorporation of radioactive leucine into yolk proteins in an in vitro bioassay consisting in the incubation of ovary fragments from the adult female shrimp, Penaeus vannamei, and immunoprecipitation of the proteins with a specific antibody to shrimp yolk protein. Thus, the SG extract from the crayfish, P. bouvieri, contains both a gonad-inhibiting hormone and a gonad-stimulating hormone (GSH), capable of acting on ovarian yolk protein synthesis in the shrimp, P. vannamei. The output of shrimp larvae spells the success of a shrimp hatchery. The induction of maturation of shrimp in captivity is done at present via eyestalk ablation of female broodstock, but this method causes increased mortality, disruption of the shrimp's endocrine system, and decreased larval viability with repeated spawnings. GSH could be used in aquaculture to increase the effect of eyestalk ablation or to substitute for this procedure completely.

Determination of taurine in biological samples by reversed-phase liquid chromatography with precolumn derivatization with dinitrofluorobenzene

A rapid and sensitive method for the determination of taurine in human milk and urine by reversed-phase liquid chromatography (LC) has been developed. It involves precolumn derivatization with dinitrofluorobenzene in NaHCO 3 buffer (pH 9.0), catalysed by dimethyl sulphoxide, separation by LC on a μBondapak Phenyl column at 40°C with acetonitrile-water (1:1) and 0.01 mol l −1 phosphate buffer (pH 5.5) as mobile phase and UV absorbance detection at 360 nm. The peak area is proportional to the concentration of taurine in the range 10–80 μg ml −1, the correlation coefficient being 0.9997. The detection limit in standard solutions is 0.01 μg ml −1 (signal-to-noise ratio = 3). The relative standard deviation ( n = 6) of retention time and peak area are 0.45% and 2.29%, respectively. The average recovery for human milk and urine are 95.8% and 101.0%, respectively.

Determination of stavudine, a new antiretroviral agent, in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A sensitive high-performance liquid chromatographic assay has been developed to determine the levels of a new antiretroviral agent, stavudine (2′,3′-didehydro-3′-deoxythymidine, d4T), in human plasma. Didanosine (2′,3′-dideoxyinosine, ddI) was used as the internal standard. The very selective sample pretreatment involved solid-phase extraction using silica gel columns. Chromatography was carried out on a μBondapak phenyl column, using a mobile phase of 0.005 M phosphate buffer (pH 6.8)—methanol (90:10, v/v) and ultraviolet detection at 265 nm. The method has been validated, and stability tests under various conditions have been performed. The detection limit is 10 ng/ml (using 500-μl human plasma samples). The bioanalytical assay has been used in a single pharmacokinetic experiment in a rat to investigate the applicability of the method in vivo.

Presence of myotropins in larval midgut extracts of lepidopteran insects: Manduca sexta, Agrotis segetum and Spodoptera exampta

Myotropic factors were found in larval midgut extracts of three lepidopteran species: Manduca sexta, Agrotis segetum and Spodoptera exempta. The myotropic activity detected in the midgut extract of M. sexta is much higher than those of the other two species. These biologically active factors were partially purified by reverse-phase C 18 Sep-pak and HPLC fractionation on a μ-Bondapak Phenyl column. A heterologous Locusta migratoria oviduct bioassay was used to monitor the myotropic activity in all purification steps. There are at least eight active HPLC fractions in M. sexta and two in both A. segetum and S. exampta, which stimulated the spontaneous contraction of the locust oviduct in vitro. The results of pronase digestion and heat treatment (100°C for 60 min) indicated that the myotropic factors present in the midgut extracts of these three species are heat-stable peptides since they were inactivated by pronase but not by boiling.

Identification, purification and initial characterization of the vitellogenesis-inhibiting hormone from the Mexican crayfish Procambarus bouvieri (Ortmann)

1. 1. By means of a single step of RP-HPLC on μBondapak-Phenyl column, a neuropeptid with vutellogenesis-inhibiting hormone (VIH) activity was isolated from a crude extract of sinus glands (SG) of the crayfish P. bouvieri. The yield was 5.4 ng/SG. 2. 2. The biological activity was tested in a heterologous in vitro bioassay as the depression of vitellogenin biosynthesis by cultured Panaeus vannamei's ovaries. 3. 3. The molecular mass was determined tobe 8388 ± 2 Da by means of mass spectrometry. 4. 4. The N-terminus was shown to be blocked by means of the dansyl-Cl-method. 5. 5. The following amino acid average composition was obtained by means of precolumn derivatization with phenylisothiocyanate: (Asx) 13, (Glx) 8, (Val) 7, (Arg, Cys, Leu) 6, (Tyr) 5, (Ala, Ile) 4, (Lys) 3–4, (Phe) 3, (Thr) 2–3, (Ser, Gly) 23, (Pro) 1. VIH consists of 72–74 amino acid residues. It does not contain Trp, His, or Met. 6. 6. The tryptic peptides of the reduced and carboxymethylated VIH and of the crustacean hyperglycemic hormone (major isoform, CHH-I) were purified on an Ultrasphere-ODS column and the peptide maps compared with respect to retention times and composition. Forty-six percent of the total amount of amino acid residues were accounted for in 5 peptides which coincided in elution time and composition. 7. 7. The remarkable resemblance between VIH and CHH-I confirms clearly that VIH belongs to the neuropeptide hormone family which includes the molt-inhibiting hormone (MIH) and the two isoforms of the crustacean hyperglycemic hormone (CHH-I and CHH-II, formerly designated CHH-B and CHH-C by us).

Rapid and sensitive method for the determination of albendazole and albendazole sulphoxide in biological fluids

A sensitive and selective reversed-phase high-performance liquid chromatographic method for the determination of albendazole and its active metabolite albendazole sulphoxide in plasma has been developed. It involves single-step extraction of plasma with dichloromethane, evaporation of the solvent and chromatography on a muBondapak phenyl column with a mobile phase of water containing 1% (v/v) triethylamine-methanol-acetonitrile (70:10:20, v/v) at pH 3.1. Run time is 12 min. The assay satisfies all of the criteria required for use in clinical pharmacokinetic studies and possesses important advantages, notably speed and expense, over current methods.

Determination of adriamycin in plasma and tissue biopsies

A simple, rapid and sensitive method for the extraction and high-performance liquid chromatographic analysis of adriamycin in tissue and plasma is described. Tissue (5-100 mg) and plasma (1 ml) samples underwent a C18 Sep-Pak extraction into methanol. Chromatography was performed on a muBondapakphenyl column using a mobile phase of acetonitrile-0.1 M ammonium formate (pH 4.0) with a flow-rate of 2 ml/min. Fluorometric detection was used with an excitation of 480 nm and an emission of 550 nm. The procedure produced a linear curve for the concentration range 25-1000 ng/ml. The development of the assay produced rapid, repeatable and accurate results for both small tissue samples and plasma.

Determination of encainide and its metabolites by high-performance liquid chromatography

Encainide (ENC) and its metabolites O-demethylencainide (ODE), 3-methoxy- O-demethylencainide (MODE), N-demethylencainide (NDE) and bis- N,O-demethylencainide (NODE) have been measured by two HPLC procedures. The method of Mayol using a μPorasil column with ethanol—water—methanesulphonic acid as mobile phase was not able to separately measure NODE and NDE in plasma. A new method is described using a μBondapak Phenyl column with acetonitrile—phosphate buffer (0.05 M, pH 7.5) that yields satisfactory separation of ENC and its metabolites. NODE was not identified as a metabolite in 23 patients analysed.

A neuropeptide with molt-inhibiting hormone activity from the sinus gland of the mexican crayfish Procambarus bouvieri (Ortmann)

1. 1. By means of reverse-phase high-performance liquid chromatography (RP-HPLC) on a μBondapak-Phenyl column, a neuropeptide with molt-inhibiting hormone (MIH) activity was isolated from a crude extract of 2000 sinus glands from the crayfish Procambarus bouvieri (Ortmann) with a yield of 25 ng/SG. 2. 2. The activity was tested in a heterology in vitro assay as the depression of ecdysteroid biosynthesis by cultured Orconectes limosus' Y-organs. 3. 3. On SDS-PAGE, the peptide migrated as a single band of approximately 6000 Da, and on IEF, it migrated as a single band to a pI of 5.50. 4. 4. The N-terminus was shown to be blocked by means of the dansyl-Cl method, while the C-terminus was shown to be isoleucine by means of carboxypeptidase-Y digestion. 5. 5. The following amino acid average composition of MIH was obtained: (Asx) 7, (Val) 6, (Glx, Leu, Arg) 5, (Cys, Lys) 4, (Ala, Ile, Tyr, Phe) 3, (Ser) 2, (Thr, Gly) 1–2, (Pro) 1. MIH has 53–55 amino acid residues and its minimal molecular mass is calculated as 6243–6402 Da. It does not contain Trp, His nor Met. 6. 6. 37.7 μg (6.3 nmol) of the molt-inhibiting hormone (MIH) and 70.7 μg (11.8 nmol) of the major peak of the crustacean hyperglycemic hormone (CHH-B) were reduced, carboxymethylated and digested with trypsin. The tryptic peptides of each of the hormones were purified on an Ultrasphere-ODS column and the peptide maps compared with respect to retention times and the composition of each peak. 7. 7. 57.4% of the total amount of amino acid residues were accounted or in five peptides which coincided in elution time and composition. 8. 8. As we have found a clear-cut difference of only two residues between MIH and CHH-B (without taking into account the amides of aspartic and glutamic acids), the present results confirm that the two hormones belong to a family of neuropeptides comprising at least one molt-inhibiting hormone and two hyperglycemic hormones. 9. 9. Evidence is presented for the existence of microheterogeneity in the structure of each of these hormones.

Single-step purification of two hyperglycaemic neurohormones from the sinus gland of procambarus bouvieri: Comparative peptide mapping by means of high-performance liquid chromatography

A crude aqueous extract from 2000 sinus glands of the Mexican crayfish Procambarus bouvieri (Ortmann) was fractionated on a μBondapak-Phenyl column. Two isoforms of the crustacean hyperglycaemic hormone, designated CHH-B and CHH-C in order of elution, were isolated in pure form. Their biochemical characterization showed a remarkable degree of homology. A tryptic digest of each isohormone was fractionated on an Ultrasphere-ODS column. Only one tryptic peptide in CHH-C was eluted later than its homologous peptide in CHH-B. On acid hydrolysis both tryptic peptides had the same composition but, as they contain Asp and Glu, we suspect that the difference resides in a double reciprocal amidation-deamidation of two acidic residues.

Determination of nalbuphine by high-performance liquid chromatography with electrochemical detection: Application to clinical samples from post-operative patients

A rapid, selective and reproducible high-performance liquid chromatographic assay with electrochemical detection was developed for the determination of nalbuphine in human plasma. The method involves extraction with chloroform—isopropanol at pH 9.4, back-extraction into dilute phosphoric acid and reversed-phase chromatography on a μBondapak phenyl column. The recovery of nalbuphine and naltrexone (internal standard) was greater than 90%. Calibration curves were linear over a concentration range of 3–36 ng/ml with coefficients of variation, within-day or between-day, not exceeding 8% at any level. Although the limit of detection was 0.3 ng/ml based on a signal-to-noise ratio of 3, the reliable limit of quantitation was 1 ng/ml (coefficient of variation 12%) using 1 ml of plasma. The dual-electrode detector was operated in the screening mode of oxidation (electrode 1, 0.3 V and electrode 2, 0.6 V), providing a greater specificity and reducing background noise. This procedure was applied to a large number of clinical samples in an intravenous dose-range pharmacokinetic study in patients.

Liquid Chromatographic Assay for Amodiaquine in Tablets and Biological Fluids

Abstract A high performance liquid chromatographic assay for quantitating amodiaquine (A) in tablets, urine, plasma, bile and saliva is described. The method involved acid extraction of the drug from tablets and chloroform extraction of its base from the biological fluids after alkalinization with ammonia. Quinidine was used as the internal standard. A μ-Bondapak phenyl column was used for separation together with a mobile phase made of methanol, water and glacial acetic acid (pH 2.3). Good chromatograms with efficient separation of drug and internal standard peaks were observed. Retention times of 5.2 and 7.1 min. were obtained for the drug and the internal standard respectively. Correlation between areas under the curve and drug concentration was high. The mean percentage recovery of A from tablets was 102.03%, while from the biological fluids, it ranged from 85.2 to 104.61%. Urine and saliva obtained from volunteers and bile obtained from animals administering amodiaquine showed chromatograms similar to those obtained for blank samples spiked with A. Interference from table't excipients of biological fluids was undetectable or negligible. The method was found to be precise and simple.

Determination of cyclodextrins in biological fluids by high-performance liquid chromatography with negative colorimetric detection using post-column complexation with phenolphthalein

A rapid and sensitive high-performance liquid chromatographic method for the analysis of β- and γ-cyclodextrin in aqueous biological fluids such as plasma, urine, or tissue homogenate is described. The chromatographic system consists of a μBondapak Phenyl column as stationary phase and a mobile phase of water with 10% methanol. After post-column addition of an alkaline solution of phenolphthalein, negative colorimetric detection is used. The elution solvent and post-column reagent were mixed in a capillary tubing of 1.5 m (1.0 mm I.D.). Two methods of sample treatment are given, one for large (1.0 ml) and one for small (0.1 ml) sample volumes. Both methods were shown to be linear and reproducible. The detection limit for β-cyclodextrin was 1.0 μg/ml (0.77 nmol/ml). The method was used in the determination of some pharmacokinetic parameters of β-cyclodextrin in rats after intravenous injection.

A neurosecretoru hyperglycemic hormone from the sinus gland of the mexican crayfish Procambarus bouvieri (Ortmann)—I. Purification and biochemical characterization of the most abundant form of the hormone

1. 1. The hyperglycemic activity of a crude extract (CE) from the sinus gland (SG) of Procambarus bouvieri (Ortmann) was recovered as a single asymmetric peak by means of reverse phase high performance liquid chromatography (RP-HPLC) on a Nova-Pak C 18 column. This peak was resolved into three symmetric peaks on rechromatography on a μBondapak-Phenyl column. Peaks were designated A, B, and C in the order of elution. Peak A had no hyperglycemic activity, while Peak B was as active as Peak C. 2. 2. Protein determination showed the following results (in ng/SG): CE—907.5, Peak A—14.5, Peak B—30.0 and Peak C—16.5. The protein content of Peaks A + B + C was 61 ng/SG, which is 6.7% of the CE protein. 3. 3. The bioassay was conducted on destalked Procambarus bouvieri. One unit of hyperglycemic activity was found to correspond to 9.4 ng of CE, 860 pg of B and 750 pg of C. 4. 4. Peak B, the most abundant form, was further characterized. On SDS-PAGE it migrated as a single band of approximately 6000 M.W. 5. 5. On I.E.F., peptide B migrated as a single band to a p I of 4.79. 6. 6. By means of the DNS-Cl method, it was shown that peptide B is N-terminally blocked. 7. 7. The carboxyl end of peptide B was found to be isoleucine by carboxypeptidase Y digestion and dansylation. 8. 8. The following amino acid average composition of peptide B was obtained from the acid hydrolysate of 2 nmoles for 20, 48 and 72 hr: (ASX) 7, (VAL) 6, (GLX) 5, (LEU, TYR, CYS, ARG) 4, (ALA, ILE, LYS) 3, (THR, SER, PRO, PHE) 2, (GLY) 1. 9. 9. Peptide B has 52 amino acid residues and its minimal M.W. was calculated as 6042 Da. It does not contain TRP, HIS or MET. 10. 10. While this work was in process, a simplified procedure utilizing only one RP-HPLC step on a μBondapak-Phenyl column was introduced. The same results as above were obtained.

High-performance liquid chromatographic assay for hydrochlorothiazide in human urine

A high-performance liquid chromatographic assay was developed for the quantitative determination of hydrochlorothiazide (HCT) in human urine. Reversed-phase separation of HCT and the internal standard, trichloromethiazide (TCMT), was accomplished on a 300 × 3.9 mm μBondapak Phenyl column. Following solvent extraction, concentrations of HCT as low as 0.25 μg/ml in urine were quantified by UV detection at 280 nm. Detector response (peak-area ratio of HCT to TCMT) was linear to 50 μg/ml. No interferences were observed in the extracts obtained from drug-free urine nor from several antihypertensive agents which are commonly co-administered with HCT. This method has been routinely employed in bioavailability studies evaluating a variety of formulations as well as characterizing the pharmacokinetics of this drug from urinary excretion data.