High-performance liquid chromatographic assay for diltiazem in small-volume blood specimens and application to pharmacokinetic studies in rats

A high-performance liquid chromatographic (HPLC) method was developed which involves the use of two 5-microns BDS silica gel columns (15 cm x 4.6 mm I.D.) in series for increased resolution and sensitivity, and an organic mobile phase for both extraction and elution of diltiazem. Plasma samples (400 microliters) were extracted using the organic mobile phase [n-hexane-methanol-dichloromethane-ammonia (370:35:30:0.3)] and the extracts were monitored at 240 nm. Desipramine (30 micrograms ml-1) was the internal standard. The limit of quantification in plasma was 20 ng ml-1 with a correlation coefficient of > or = 0.999 within the 20-800 ng ml-1 standard window. The inter- and intra-assay R.S.D.s were within 5%. The recovery of diltiazem varied from 101.1% at 20 ng ml-1 to 93.7% at 400 ng ml-1. The method was applied to the investigation of diltiazem absorption in a rat. Drug absorption was based on the intestinal single-pass perfusion model. The concentration of diltiazem in all test perfusion solutions was 1 mg ml-1 (2.4 mM) and the flow-rate through the system was 3.33.10(-3) ml s-1. A non-specific mucolytic absorption enhancer was also added to a diltiazem solution and studied in the in situ system. The pharmacokinetics of diltiazem hydrochloride were investigated in two study groups of Wistar rats (n = 4). A two-sample Student's t-test was employed to compare values of the area under the curve (AUC). The pharmacokinetic data indicated that the AUC in the group which received the enhancer [18.12 +/- 5.43 ng ml-1 h-1 (+/- S.D.)] was higher than that in the control group (11.49 +/- 3.67 ng h-1 ml-1), t-test; p = 0.0483. Hence it was shown that administration of an enhancer could increase the oral bioavailability of diltiazem.     

High-performance liquid chromatography-mass spectrometry for the quantification of nortriptyline and 10-hydroxynortriptyline in plasma

A highly sensitive and selective method for the quantification of nortriptyline and its major 10-hydroxy metabolite in plasma is described. The method is based on liquid-liquid extraction in combination with acid dehydration of the 10-hydroxy metabolite to the less polar 10,11-dehydronortriptyline. Deuterium labelled internal standards ([2H4]NT and [2H3]10-OH-NT) were used and the compounds were separated by reversed-phase HPLC and detected using atmospheric pressure chemical ionisation and mass spectrometry. The limit of quantification was 0.8 ng/ml for both compounds. A 1-ml volume of plasma was used for analysis in the concentration range 0.8-32 ng/ml. The within- and between-day coefficients of variation were 11% in the low, 1.6 ng/ml range, and 7% at 8 ng ml/ml. Using this method it was possible to quantify plasma concentrations for 168 h following a single oral dose of 25 mg of nortriptyline with good accuracy and precision.     

Application of liquid chromatography-mass spectrometry(n) analyses to the characterization of novel glyburide metabolites formed in vitro

Up to date five different staphylococcal lipase genes, two of Staphylococcus aureus (sal-1 and sal-2), two of Staphylococcus epidermidis (sel-1 and sel-2) and one of Staphylococcus hyicus (sh1) have been cloned and sequenced. All corresponding proteins are organised as pre-pro-enzymes: the pre-region represents the signal peptide, the pro-region has a length between 207 and 267 amino acids, and the mature part comprises 380 to 400 amino acids. We found that the lipases are secreted in the pro-lipase form. The processing of the pro-form to the mature enzyme occurs extracellular by a specific protease. Interestingly the pro-lipase reveals not much less activity compared to the mature lipase. There are evidences that the pro-region acts as an intramolecular chaperone which facilitates translocation not only of the native lipase but also of a number of completely unrelated proteins fused to the pro-peptide. It was also observed that the pro-region protects the proteins from proteolytic degradation. While the Staphylococcus aureus and Staphylococcus epidermidis lipases have only lipase (esterase) activity, the related Staphylococcus hyicus enzyme (SHL) is distinguished by both lipase and phospho-lipase activity. The biochemical and catalytic properties of these lipases are described in the accompanying article (Simons, J.W., G?tz, F., Egmont, M.R. and Verheij, H.M., 1998. Staphylococcal lipases: Biochemical properties. Accompanying article).     

High-performance liquid chromatographic analysis of isoniazid and its dosage forms

A high-performance liquid chromatographic analysis is described for isoniazid as a drug entity and in its tablet and injectable dosage forms. After incorporation of the drug or dosage form in a solvent mixture and addition of an internal standard, tribenzylamine, an aliquot is chromatographed using a pellicular silica gel medium followed by UV spectrophotometric detection at 254 nm. The response of the chromatographic system was linear over a concentration range corresponding to 20-200% of the labelled amount of isoniazid. Comparison of the results with those obtained by the official USP XIX method indicates similar accuracy and precision. The advantages of the proposed method are its simplicity and rapidity, its potential for automation, and its specificity. The specificity was demonstrated in the presence of potential degradation products of isoniazid, other drugs used with isoniazid in combination dosage forms, and an adduct formed by the reaction of isoniazid with lactose in the tablet.     

Coupled-column liquid chromatographic analysis of epirubicin and metabolites in biological material and its application to optimization of liver cancer therapy

A specific, sensitive and fully automated coupled-column LC method for the determination of the anthracycline cytostatic epirubicin and four metabolites in the biological materials human plasma, liver homogenate and liver tumour homogenate has been developed. System-integrated sample processing was achieved using a new restricted access silica precolumn packing. This porous Alkyl-Diol Silica (ADS) was specially designed for the direct and repetitive injection of proteinaceous samples. It consists of a hydrophilic and electroneutral external particle surface (glyceryl-residues) and a hydrophobic reversed-phase internal surface (butyryl-, octanoyl- or octadecyl-residues). These bimodal chromatographic properties allow retention of low molecular analytes by classical RP-chromatography exclusively at the lipophilic pore surface. Macromolecular constituents of the sample matrix (e.g. proteins) are size-excluded by 5 nm pores and quantitatively eliminated in the interstitial void volume. On-line analysis was performed by coupling a C4-Alkyl-Diol precolumn (20 x 4 mm i.d., particle size 25 microns) and LiChrospher RP Select B analytical column (250 x 4 mm i.d., particle size 5 microns) via an electrically driven six-port valve. Separation of the parent compound and its metabolites was achieved with a mobile phase consisting of water (0.1% triethylamine, v/v, pH 2.0 adjusted with trichloroacetic acid)-acetonitrile (70:30, v/v) at a flow rate of 1 ml min-1. The analytes were detected using their natural fluorescence (excitation 445 nm, emission 560 nm). The method described is used for the determination of pharmacokinetics of epirubicin and its metabolites in order to evaluate and optimize treatment regimen of liver cancer chemoembolization therapy.     

Use of liquid chromatography-mass spectrometry in the analysis of residues of antibiotics in meat and milk

The advent of affordable LC-MS systems has led to a massive increase in a number of publications describing quantitative methods for the analysis and confirmation of veterinary drug residues. The lack of volatility and thermal instability of many antibiotics makes LC-MS the method of choice for their analysis. In the review, analytical methods for the determination of residues of each of the major classes of antibiotics are presented.     

A pharmacodynamic study of morphine and its glucuronide metabolites after single morphine dosing in cancer patients with pain

Our previous results showed that hematopoietic stem cells from 16-week-old Sl/Sl(d) mice are not as competitive as congenic +/+ control stem cells. Possible explanations for these findings are that the Steel stem cells are either inherently defective or lose competitive ability by residence in an environment lacking membrane-bound Steel factor. In the present report, any long-term effects of the Steel microenvironment were eradicated by transferring neonatal Sl(d)/Sl(d) marrow and spleen cells into an irradiated but otherwise normal adult hematopoietic microenvironment. Host cells were completely replaced by donor cells within 6 weeks. Eight months after transplantation, the Sl(d)/Sl(d) and similarly treated +/+ littermate control cells from the primary recipient marrow were competed against genetically marked normal cells in an irradiated secondary host. The Steel cells were as competitive as the control cells demonstrating that Steel stem cells are not inherently defective. Results suggest that the stem cells, when retained in the mutant environs into adulthood, are either reduced in number or phenotypically altered by lack of the membrane-bound Steel factor.     

Determination of sedatives and anesthetics in plasma by liquid chromatography-mass spectrometry with a desalting system

Though liquid chromatography-mass spectrometry (LC-MS) is a powerful tool for analysis of drugs and their metabolites, it does not allow the use of a non-volatile buffer such as phosphate buffer. We used a column-switching desalting system in combination with atmospheric pressure chemical ionization LC-MS for analysis of sedatives and anaesthetics. The drugs examined were flumazenil, butorphanol, midazolam, lorazepam, phenobarbital and flunitrazepam. The separation was carried out on a reversed-phase column using acetonitrile-0.1 M phosphate buffer as a mobile phase. The mass spectra are almost the same as those obtained by direct analysis and the molecular ions were clearly observed. In the analysis, phosphate buffer was completely removed with the trapping column and did not interfere with ionization of the drugs in MS. The chiral separation of lorazepam was achieved on a chiral column with UV, optical rotatory detection and MS. This method is sufficiently sensitive and accurate for the pharmacokinetic studies of these drugs in biological samples.     

High-performance liquid chromatographic method for the determination of norfloxacin glutamate and glucuronate in solid and liquid dosage forms and its application to stability testing

A simple, precise, stability-indicating reversed-phase high-performance liquid chromatographic method for norfloxacin glutamate and norfloxacin glucuronate in liquid and solid dosage forms is described. Chloronitrodiazepine was used as the internal standard. The eluent used with a C18 bonded phase column was methanol-water-diethylamine (50:50:0.4, v/v/v) (pH* 5.5). The effects of the eluent pH*, the ratio of methanol to water, and the quantity of diethylamine on the retention times of the sample and internal standard were investigated. The method showed good linearity in the range 1-45 micrograms ml-1 for norfloxacin. Solid samples were ground, dissolved in the eluent, filtered, and then determined by this method. Liquid samples were dissolved in the same solvent. The average recoveries of norfloxacin glutamate and norfloxacin glucuronate in their simulated preparations were 99.5% for solid products and 99.8% for liquid products. The method was applied to the study of the thermal stability of the drugs by following the degradation of norfloxacin glutamate and glucuronate in the four products in accelerated tests at 37-80 degrees C for up to 3 months. Shelf-lives at 25 degrees C of the four products were predicted from the results assuming zero- and first-order kinetics of decomposition, and were at least 1.5 years for liquid products and 2 years for solid products.     

Selection of phenytoin responders and nonresponders in male and female amygdala-kindled Sprague-Dawley rats

PURPOSE: We recently described that, by repeated testing of the anticonvulsant phenytoin (PHT), it is possible to select responders and nonresponders from large populations of amygdala-kindled Wistar rats. Whereas responders show marked and reproducible increases of focal seizure threshold (afterdischarge threshold: ADT) on repeated testing of PHT, 75 mg/kg i.p., nonresponders do not show any significant ADT increase after this dose, thus allowing use of these subgroups in the search for mechanisms of pharmacoresistance in temporal lobe epilepsy. In this study, we examined whether PHT responders and nonresponders can also be selected from large groups of kindled rats of the Sprague-Dawley strain. METHODS: Male and female Sprague-Dawley rats were amygdala kindled, followed by once weekly i.p. testing of PHT. RESULTS: In contrast to recent experiments in Wistar rats, 75 mg/kg PHT did not induce significant ADT increases in Sprague-Dawley rats, indicating strain differences in response to this drug after kindling. When the dose was lowered to 50 or 25 mg/kg, significant and reproducible ADT increases were obtained in Sprague-Dawley rats of both genders. Therefore these doses were used for selection of responders and nonresponders in a total of 42 rats. Almost 50% of the rats were PHT responders, whereas no rat was a nonresponder when tested in up to six subsequent drug trials. Many rats were variable responders (i.e., showed ADT increases in some but not all trials), which was not due to low or variable drug absorption after i.p. injection. CONCLUSIONS: The data indicate that, in contrast to Wistar rats, Sprague-Dawley rats are not suited for selection of PHT nonresponders, but rather are quite responsive to this drug. A further difference to the Wistar strain is the truncated dose-response with loss of anticonvulsant efficacy at 75 mg/kg in kindled Sprague-Dawley rats, which may, at least in part, explain the inconsistent results reported on the anticonvulsant efficacy of PHT in this strain in the literature. The lack of anticonvulsant activity after administration of 75 mg/kg may be a result of kindling, because administration of this dose before kindling causes a significant ADT increase in this strain. This kindling-induced alteration of the anticonvulsant activity of PHT is a phenomenon that contrasts Sprague-Dawley with Wistar rats and deserves further investigation.     

Liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry of omega- and (omega-1)-hydroxylated metabolites of elaidic and oleic acids in human and rat liver microsomes

A peptide that inhibits the human cholesteryl ester transfer protein (CETP) was isolated from hog plasma by ultracentrifugation, two sequential column chromatographies and electroelution from gels. Molecular weight of the peptide was determined to be approximately 3 kDa on the SDS-PAGE. The peptide contained 28 amino acids with an identical sequence to the amino terminus of hog apolipoprotein-CIII except two amino acid residues: -Pro-Glu- at the fifth and sixth amino acids from the amino terminus in the isolated peptide, in contrast to -Leu-Leu- in hog apo-CIII. A peptide synthesized chemically according to the amino acid sequence of the peptide (designated P28) showed approximately the same degree of CETP inhibitory activity as the isolated peptide. Synthetic peptides with different number of amino acids were also tested for CETP inhibition. Among the peptides, the one with 20 amino acid residues (P20) from the amino terminus showed the highest inhibitory activity against the CETP. The peptide appeared to be associated with the hog high-density lipoproteins (HDL), as determined by immunoblot analysis using antibody against P28. The CETP-inhibitory activity of the peptide was examined in vivo using diet-induced hypercholesterolemic rabbits. When the peptide was injected into the rabbits (7-9 mg/kg body weight), approximately 75% CETP activity disappeared from the plasma in 1 h after the injection and the effect lasted up to 30 h. The inhibition of CETP in vivo led to a concomitant decrease in total plasma cholesterol level up to 30% and an increase in the level of HDL-cholesterol up to 32%. The cholesterol concentrations in the rabbit plasma gradually recovered to the initial level after 48 h.     

Purification and characterization of a recombinant hepatitis E protein vaccine candidate by liquid chromatography-mass spectrometry

A protein with a molecular mass of approximately 62.10(3), derived from open reading frame 2 (ORF-2) of the hepatitis E virus (HEV: Burma strain), was expressed in a baculovirus expression vector and purified to homogeneity. The recombinant 62 kDa protein appeared to be a doublet, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Tryptic digestion in conjunction with laser desorption mass spectrometry (LD-MS) and sequence analysis of the tryptic peptides indicated that the amino terminus was blocked, although no proteolytic degradation occurred. The determined internal sequences of peptides were in agreement with the predicted ORF-2 protein. Reversed-phase liquid chromatography coupled to electrospray mass spectrometry (LC-MS) resolved the doublet proteins into two major components with molecular masses of 56548.5 and 58161.4. Confirmation of the amino terminus of the molecule by LD-MS post-ion decay enabled us to tentatively assign the carboxyl terminus of each species at residues 540 and 525. Sequencing of the intact protein by automated carboxyl terminal sequencing confirmed that the carboxyl terminus was truncated and that the sequence assignment predicted by LC-MS was correct.     

Analysis of novel hydroperoxides and other metabolites of oleic, linoleic, and linolenic acids by liquid chromatography-mass spectrometry with ion trap MSn

Linoleate is oxygenated by manganese-lipoxygenase (Mn-LO) to 11S-hydroperoxylinoleic acid and 13R-hydroperoxyoctadeca-9Z,11E-dienoic acid, whereas linoleate diol synthase (LDS) converts linoleate sequentially to 8R-hydroperoxylinoleate, through an 8-dioxygenase by insertion of molecular oxygen, and to 7S,8S-dihydroxylinoleate, through a hydroperoxide isomerase by intramolecular oxygen transfer. We have used liquid chromatography-mass spectrometry (LC-MS) with an ion trap mass spectrometer to study the MSn mass spectra of the main metabolites of oleic, linoleic, alpha-linolenic and gamma-linolenic acids, which are formed by Mn-LO and by LDS. The enzymes were purified from the culture broth (Mn-LO) and mycelium (LDS) of the fungus Gaeumannomyces graminis. MS3 analysis of hydroperoxides and MS2 analysis of dihydroxy- and monohydroxy metabolites yielded many fragments with information on the position of oxygenated carbons. Mn-LO oxygenated C-11 and C-13 of 18:2n-6, 18:3n-3, and 18:3n-6 in a ratio of approximately 1:1-3 at high substrate concentrations. 8-Hydroxy-9(10)epoxystearate was identified as a novel metabolite of LDS and oleic acid by LC-MS and by gas chromatography-MS. We conclude that LC-MS with MSn is a convenient tool for detection and identification of hydroperoxy fatty acids and other metabolites of these enzymes.     

Determination of medetomidine, atipamezole and midazolam in pig plasma by liquid chromatography-mass spectrometry

Medetomidine, atipamezole and midazolam in pig plasma were determined by liquid chromatography-mass spectrometry (LC-MS) with an atmospheric pressure chemical ionization interface system by the use of detomidine as an internal standard. The method was applied to studies of pharmacokinetic behaviour of these drugs.     

Effects of acute inhalation exposure to isoamyl nitrite on the hypothalamo-pituitary-adrenal axis in male Sprague-Dawley rats

Isoamyl nitrite (IAN) is a member of the family of volatile organic nitrites that exert vasodilatory effects and have recently exhibited a considerable potential for inhalation abuse. In an effort to provide mechanistic insight into the neurotoxic effects and abuse potential of these agents, the present study was designed to evaluate the acute effects of IAN on the hypothalamo-pituitary-adrenal (HPA) axis. Attempts were also made to correlate the neuroendocrine effects of IAN with its pharmacokinetic profile. Male Sprague-Dawley rats were exposed to 600 or 1200 ppm IAN by inhalation for 10 or 30 min. Following exposure, adrenocorticotropic hormone (ACTH) and corticosterone in plasma and corticotropin-releasing factor (CRF) in three brain regions (hypothalamus, hippocampus, and frontal cortex) were determined by radioimmunoassay. Levels of IAN in the three brain regions as well as in blood were measured by gas chromatography to determine the target tissue concentrations responsible for neuroendocrine changes. Uptake of IAN into blood and all brain regions was very rapid, as stable concentrations were achieved within 10 min of exposure and maintained for 30 min of continuous inhalation. Plasma corticosterone decreased significantly after 10 min inhalation of both IAN doses, and returned to control levels after 30 min. Moreover, plasma ACTH was significantly increased by 10 and 30 min of exposure to 600 and 1200 ppm IAN, while hypothalamic CRF increased significantly after 30 min of exposure to the 600 ppm dose. These latter findings suggest activation of the hypothalamus and pituitary due to a reduction in negative feedback resulting from the initial decrease in corticosterone. Although plasma ACTH was greatly increased after 30 min, plasma corticosterone levels were unchanged, indicating that IAN primarily acts to inhibit the synthesis or secretion of adrenal steroids and that activation of the HPA axis is not involved in the behavioral manifestations of IAN inhalation. These compensatory effects of HPA axis regulation, and possibly the vasodilatory properties of IAN, also likely precluded the establishment of definitive relationships between observed changes in hormone levels and blood or regional brain concentrations of the inhalant.     

Immunoaffinity extraction of 4-hydroxy-2-(4-methylphenyl)benzothiazole and its metabolites for determination by gas chromatography-mass spectrometry

Immunoaffinity extraction of 4-hydroxy-2-(4-methylphenyl)benzothiazole and its metabolites, together with the corresponding meta-isomers has been achieved by the use of an antibody raised against an immunogen, an O-carboxymethyloxime-bovine serum albumin conjugate of 4-hydroxy-2-(4-formylphenyl)benzothiazole. The antibody produced exhibited a broad spectrum of affinity, not only for metabolites oxidized at the 4-methyl group of the benzene moiety but also for the corresponding meta-isomers. Up to 4 micrograms in total of these benzothiazoles could be extracted on the immunoaffinity adsorbent and recovered almost quantitatively by elution with 90% methanol. The resulting chromatogram was free from any interference. The eluted compounds were derivatized by conversion to their methyl esters and/or trimethylsilyl ethers, and subsequently separated into individual benzothiazoles by means of gas chromatography-mass spectrometry. The derivatized compounds were monitored using a characteristic ion, [M-CH3]+., and the limit of detection was 10 fmole. The peak height ratio of each metabolite to its corresponding meta-isomer internal standard was plotted against the concentration of the former and good linearity was observed over the range 0.2-5 ng/ml.     

Measurement of tacrolimus (FK506) and its metabolites: a review of assay development and application in therapeutic drug monitoring and pharmacokinetic studies

Tacrolimus (FK506, Prograf) is a macrolide immunosuppressant used for the prevention of organ rejection after transplantation. Tacrolimus demonstrates considerable interindividual variation in its pharmacokinetic profile. This has caused difficulty in defining the optimum regimen and has highlighted the need for therapeutic drug monitoring. Several assay methods for the measurements of tacrolimus in biological specimens have been developed. These assay methods were used for therapeutic drug monitoring and/or pharmacokinetic studies. Two commercially available immunoassays, based on the same monoclonal antibody to tacrolimus, have been used for therapeutic drug monitoring of tacrolimus in whole blood. For pharmacokinetic studies, the assay methods were used to measure tacrolimus and its metabolites in very low concentrations in selected biological matrixes to determine the metabolic and pharmacokinetic profiles of this drug.     

Inhalation toxicokinetics of butoxyethanol and its metabolite butoxyacetic acid in the male Sprague-Dawley rat

A total of 16 male Sprague-Dawley rats were continuously exposed to 20 ppm or 100 ppm butoxyethanol (BE) vapor for 1, 2, 3, 4, 6, 8, 10, or 12 days. Urine was collected in 24-h intervals and stored at -70 degrees C. At the end of the exposure the animals were euthanized by decapitation and tissue samples of blood, muscle, liver and were rapidly collected and frozen to -70 degrees C. The samples were later derivatized and analyzed for BE and its major metabolite butoxyacetic acid (BAA) by electron capture gas chromatography. BE and BAA were rapidly distributed to the tissues examined. The concentration of BE in blood was slightly higher, and that of BAA markedly higher than in other tissues, indicating weak (BE) and pronounced (BAA) blood protein binding, respectively. BE was efficiently metabolized and the blood clearance averaged 2.6 l/h per kg, corresponding to a hepatic extraction ratio of about 0.75. The renal clearance of BAA (average 0.53 l/h per kg) corresponded to approximately 15% of the renal blood flow. The kinetics of BE and BAA were linear up to 100 ppm. There were no clear indications of changes in the toxicokinetics, such as metabolic induction or inhibition of metabolism or excretion, during the course of the exposure. The recovery of BAA in urine was 64% of the calculated inhaled amount of BE, on an equimolar basis.     

Comparative metabolism and disposition of gemfibrozil in male and female Sprague-Dawley rats and Syrian golden hamsters

Antibodies (Abs) can contribute to the cure of a viral infection, in principle, in two ways by: (1) binding to infected cells and thereby reducing the production of progeny virus [here termed cell-targeting (CT) activity] and (2) reacting with released progeny virus and thereby inhibiting the spread of the infection [termed virus neutralizing (VN) activity]. We have previously shown that a pulmonary influenza virus infection in severe combined immunodeficient mice could be cured by treatment of these mice with hemagglutinin (HA)-specific monoclonal Abs (mAbs) that mediated both of the above activities. Although the therapeutic activity of these mAbs correlated with their VN activity, it remained unclear how much their CT activity contributed to the Ab-mediated recovery process. To clarify this point, we tested the therapeutic efficacy of two mAbs of IgG2a isotype that mediated CT but no VN activity: one specific for the viral neuraminidase and the other for matrix protein 2. Both mAbs reduced pulmonary virus titers by 100- to 1000-fold but they failed to clear the infection, even when administered in combination and at therapeutically saturating concentrations. The results suggest that CT activity contributes significantly also to the therapeutic activity of HA-specific mAbs and further support the notion that VN-activity is required for Ab-mediated virus clearance.