High-performance liquid chromatography determination of polydeoxyribonucleotides in plasma: its application to the determination of defibrotide's pharmacokinetics in the rabbit.

We describe an HPLC method for the determination of whole polydeoxyribonucleotides in animal plasma. This method was compared to a colorimetric method, which evaluates the sugar moiety of polydeoxyribonucleotides, and to an agarose gel electrophoresis method, which evaluates the whole polydeoxyribonucleotides as does the HPLC method, and was found to give results very close to those obtained with these two other methods. A pharmacokinetic study of the antithrombotic, profibrinolytic, polydeoxyribonucleotidic drug defibrotide was carried out by evaluating the plasma drug levels by these three methods. The pharmacokinetic parameters calculated from the data are very similar.     

Biological spin trapping II. Toxicity of nitrone spin traps: dose-ranging in the rat

To obtain the strongest possible free radical spin adduct signal using the electron paramagnetic resonance spectroscopy-spin trapping technique, it is desirable to load an animal with the highest dose of spin trap possible. One hundred and twenty six male Sprague-Dawley rats were used to establish the toxic dose range for PBN (-phenyl N-tert butyl nitrone) and 18 other similar spin traps. The lethal dose of PBN was found to be approximately 100 mg/100 g BW (0.564 mmol/100 g). The 18 other compounds were then tested, and their toxicities were gauged in terms of molar equivalents to PBN. Of these spin traps, DMPO (5,5-dimethyl-1-pyrroline-N-oxide) was found to be the least toxic (no toxic signs at twice the lethal dose for PBN) while 2,6-difluoro-PBN and M₄PO (3,3,5,5-tetramethyl-1-pyrroline-N-oxide) were the most toxic, both causing death at one eighth the PBN-equivalent lethal dose. Nine of the 18 nitrones appeared non-toxic at the 0.25 PBN-equivalent lethal dose level.     

Unique in vivo applications of spin traps

The ultimate goal of in vivo electron spin resonance (ESR) spin trapping is to provide a window to the characterization and quantification of free radicals with time within living organisms. However, the practical application of in vivo ESR to systems involving reactive oxygen radicals has proven challenging. Some of these limitations relate to instrument sensitivity and particularly to the relative stability of these radicals and their nitrone adducts, as well as toxicity limitations with dosing. Our aim here is to review the strengths and weaknesses of both traditional and in vivo ESR spin trapping and to describe new approaches that couple the strengths of spin trapping with methodologies that promise to overcome some of the problems, in particular that of radical adduct decomposition. The new, complementary techniques include: (i) NMR spin trapping, which monitors new NMR lines resulting from diamagnetic products of radical spin adduct degradation and reduction, (ii) detection of *NO by ESR with dithiocarbamate: Fe(II) "spin trap-like" complexes, (iii) MRI spin trapping, which images the dithiocarbamate: Fe(II)-NO complexes by proton relaxation contrast enhancement, and (iv) the use of ESR to follow the reactions of sulfhydryl groups with dithiol biradical spin labels to form "thiol spin label adducts," for monitoring intracellular redox states of glutathione and other thiols. Although some of these approaches are in their infancy, they show promise of adding to the arsenal of techniques to measure and possibly "image" oxidative stress in living organisms in real time.     

High-performance liquid chromatography of coproporphyrin isomers.

A reversed-phase system is described for the simultaneous isocratic separation of coproporphyrin I, II, III and IV isomers. The retention behaviour of coproporphyrin I and III is studied in detail. The method is suitable for both analytical and semi-preparative separation.     

High-performance liquid chromatographic analysis of staurosporine in vivo: Its translocation and pharmacokinetics in rats

The protein kinase inhibitor staurosporine (Stsp) has been used extensively to study physiological functions, biochemical mechanisms, and cancer therapy. Using an HPLC assay for Stsp developed in our laboratory, we find that only 0.7% of Stsp remains in circulating blood of rats 5 min after injection. In vitro, Stsp is adsorbed to red blood cells (RBC) weakly and reversibly. In vivo, all but 1.2–2.5% of Stsp injected is adsorbed by the heart and lungs in one passage through them, indicating that the endothelium acts as a major Stsp sink. Following initial adsorption, pharmacokinetic studies demonstrated that Stsp had a half-life of 51.6 min in plasma and 75.3 min in RBC. Thus, plasma Stsp was in the cancer therapy range of 1–10 ng/ml for 2.7 h following a bolus injection. This data indicates that a bolus injection of Stsp must be followed by a continuous infusion of low Stsp concentration for several days to produce the G1 arrest in cells necessary to stop cell proliferation.     

High-performance liquid chromatographic assay of indomethacin and its application in pharmacokinetics in healthy volunteers

A new sensitive high-performance liquid chromatographic method for indomethacin from plasma on a reversed-phase column (C_1$$$) has been developed. The method involves precipitation of plasma with perchloric acid followed by diethyl ether extraction. The assay is quantitative down to 0.25 μg ml⁻¹ from a 200-μl aliquot of plasma with a detection limit of 0.1 μg ml⁻¹ and a recovery of approximately 90%.

The method was applied to single-dose studies with volunteers under various dietary restrictions. The results of these studies indicated that intrasubject variability within these regimens may be as important a factor as the intersubject variability already documented for this drug. These results have important implications in the determination of bioavailability and pharmacokinetic parameters of this drug.     

High-performance liquid chromatography for the determination of 3-n-butylphthalide in rat plasma by tandem quadrupole mass spectrometry: application to a pharmacokinetic study

A rapid, sensitive and specific high performance liquid chromatography-electrospray ionization tandem quadrupole mass spectrometry (HPLC-MS/MS) method was developed and validated for the determination of 3-n-butylphthalide in rat plasma. Following protein precipitation with acetonitrile, 3-n-butylphthalide and glipizide (internal standard, I.S.) were separated using a gradient elution program on a C18 column and detected by mass spectrometry in positive ion mode with the multiple reaction monitoring (MRM) mode using the respective precursor to product ion combinations of m/z 191/145 for 3-n-butylphthalide and m/z 446/321 for glipizide, respectively. The total chromatographic running time was 2.5 min. The method was linear over the concentration range of 11.14-3480.00 ng/mL, using as little as 100 microL plasma. The lower limit of quantification (LLOQ) was 5.57 ng/mL. Finally, the method was successfully used to support a preclinical pharmacokinetic study of 3-n-butylphthalide in rats following intravenous administration.     

High-performance liquid chromatography for the purification of restriction endonucleases, application to BanII, SacI, and SphI

Conventional fractionation methods are time consuming, thus they prolong the time required to process low-stability restriction enzymes. We now report a rapid and effective two-step chromatographic method that affords high purity endonucleases in a short time. Accordingly, an inexpensive chromatographic adsorbent such as phosphocellulose or dyed agarose in the first step is coupled to a high-performance ion exchanger, namely, MonoQ, in the second step. The purification schemes reported here are now in routine use to prepare high-purity BanII, SacI, and SphI as judged by the "overdigestion" and "cut-ligate-recut" stringent quality tests.     

High-performance liquid chromatography of lipids for the identification of human metabolic disease.

We describe a system for quantitative lipid analysis employing ternary gradient high-performance liquid chromatography with evaporative light scattering detection. This technique was applied to extracts of cultured fibroblasts, cultured lymphocytes, and leukocytes and to liver and spleen biopsy specimens. Separation of nonpolar lipids, glycolipids, phospholipids, and sphingolipids was achieved in a single run. Detection did not depend on the presence of any specific chemical reactions, uv absorption, or fluorescence. The sensitivity of the technique is well below 200 ng for individual lipids, and many individual lipid classes were detected in samples as small as 1 mg of total protein, the yield of a single flask of cultured skin fibroblasts. The characteristic stored lipids cholesterol ester and sphingomyelin were seen in excess in human fibroblast cultures from patients with Wolman's disease and Niemann-Pick disease, respectively. A biopsy spleen sample from a patient with Gaucher's disease showed a large glucosylceramide peak. This system provides a tool for detecting lipids that accumulate in tissues of patients with currently unidentified metabolic storage disorders.     

High-performance liquid chromatography of type-III heptocarboxylic porphyrinogen isomers.

A reversed-phase h.p.l.c. system is described for the separation of the four type-III heptacarboxylic porphyrinogen isomers. The effects of buffer concentration, pH and type and proportion of organic modifier in the mobile phase on retention and resolution of isomers were studied. Optimum separation on an ODS-Hypersil column was by elution with a ternary mobile phase of acetonitrile, methanol and 1 M-ammonium acetate, pH 5.16 (7:3:90, by vol.). Isomer identification was based on a comparison of their retention times with those of authentic standards, and was further confirmed by h.p.l.c. analysis of the characteristic mixture of three pentacarboxylic porphyrins formed after partial decarboxylation of individual isomers in 0.3 M-HCl at 160 degrees C.     

High-performance liquid chromatography with mercury cathode electrochemical detection: application to lipid hydroperoxide analysis

Lipid hydroperoxide species can be analyzed with high sensitivity and specificity, using reversed-phase high-performance liquid chromatography with reductive mode electrochemical detection on a mercury drop cathode [HPLC-ED(Hg)]. The purpose of this study was to examine different variables in the operation of HPLC-ED(Hg) and to select optimal conditions for the analysis of several biologically relevant peroxides, including species derived from cholesterol, cholesteryl linoleate, oleate, linoleate, and two synthetic phosphatidylcholines. Parameters such as operating potential and mobile-phase solvent proportions, electrolyte composition, and ionic strength were evaluated for each peroxide class. Under optimal conditions, we have achieved baseline separation of four cholesterol hydroperoxide species, not only from one another, but also from phospholipid hydroperoxides; detection limits were <0.3 pmol and <30 pmol for the cholesterol and phospholipid hydroperoxides, respectively.     

Sonochemistry of nitrone spin traps in aqueous solutions. Evidence for pyrolysis radicals from spin traps

When argon-saturated aqueous solutions of alpha-phenyl-N-tert-butylnitrone (PBN) were sonicated, the spin adducts PBN-Phenyl (Ph), PBN-X, and PBN-H were observed. It can be inferred that PBN-Ph and -X arise from spin adducts of thermal decomposition products of PBN induced by the high temperature due to ultrasonic cavitation. The ESR signal of PBN-H was observed at a lower PBN concentration than those of PBN-Ph and PBN-X. The ratios of ESR intensity of PBN-H to those of PBN-Ph and PBN-X increased with the final temperatures of the cavitation bubbles created by different rare gases. The spin adducts of methyl and tert-butyl radicals from the pyrolysis of PBN, induced by the high temperatures due to cavitation, were found from spin trapping experiments in which 3,5-dibromo-2,6-dideuterio-4-nitrosobenzene sulfonate was used as a spin trap. Similar spin adducts induced by pyrolysis were also observed in sonicated aqueous solutions of other nitrone spin traps, such as alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone, and alpha-(4-nitrophenol) N-tert-butylnitrone. The greater the hydrophobicity of the spin traps, as measured by the 2-octanol/water partition coefficients, the lower the concentration of spin trap at which methyl radicals generated by thermal decomposition of the spin trap can be observed. The present results indicate that the nonvolatile, highly hydrophobic spin traps accumulate preferentially in the interfacial region of cavitation bubbles where they undergo thermal decomposition during cavitation to produce the radicals.     

In vivo spin trapping of nitric oxide from animal tumors.

Spin trapping/electron paramagnetic resonance (EPR) spectroscopy allows specific detection of nitric oxide (NO) generation, in vivo. However, in order to detect an EPR signal in living organism, usually a stimulation of immune system with LPS is used to achieve higher than physiological NO levels. Here, we report non-invasive spin trapping of NO in tumors of non-treated, living animals. EPR spectroscopy was performed at S-band to detect NO in Cloudman S91 melanoma tumors growing in the tail of living, syngeneic hosts-DBA/2 mice. Iron (II) N-(dithiocarboxy)sarcosine Fe2+(DTCS)(2) was used as the spin trap. The results were confirmed by X-band ex vivo study. A characteristic three-line spectrum of NO-Fe(DTCS)(2) (A(N)=13 G) was observed (n=4, out of total n=6) in non-treated tumors and in tumors of animals treated with l-arginine. Substrate availability did not limit the detection of NO by spin trapping. Half-life time of the NO-Fe(DTCS)(2) in tumor tissue was about 60 min. The feasibility of non-invasive spin trapping/EPR spectroscopic detection of NO generated in tumor tissue in living animals, without additional activation of the immune system, was demonstrated for the first time.     

Determination of dicentrine in rat plasma by high-performance liquid chromatography and its application to pharmacokinetics

A simple high-performance liquid chromatographic method was developed to study the pharmacokinetics of dicentrine in rat plasma after 10 mg/kg intravenous administration. After addition of an internal standard (coumarin), plasma was deproteinized by acetonitrile for sample clean-up. The drugs were separated on a reversed-phase Nucleosil C₁₈ column (250 × 4 mm I.D., particle size 5 μm) and detected by photodiode-array detection at a wavelength of 308 nm. Acetonitrile-water (35:65, v/v, pH 2.5–2.8, adjusted with orthophosphoric acid) was used as the mobile phase. A biphasic phenomenon with a rapid distribution followed by a slower elimination phase was observed from the plasma concentration-time curve.     

High-performance liquid chromatography method for the quantification of entacapone in human plasma

A simple, sensitive and selective HPLC method with UV detection (315 nm) was developed and validated for quantitation of entacapone in human plasma, the newest addition to the group of antiparkinsonian agents. Following a single-step liquid-liquid extraction (LLE) with ethyl acetate/n-hexane (30/70, v/v), the analyte and internal standard (rofecoxib) were separated using an isocratic mobile phase of 30 mM phosphate buffer (pH 2.75)/acetonitrile (62/38, v/v) on a reverse phase C18 column. The lower limit of quantitation was 25 ng/mL, with a relative standard deviation of less than 8%. A linear range of 25-2500 ng/mL was established. This HPLC method was validated with between-batch and within-batch precision of 2.2-4.2% and 1.7-7.8%, respectively. The between-batch and within-batch accuracy was 98.7-107.5% and 97.5-106.0%, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of entacapone in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method is sensitive, simple and repeatable enough to be used in pharmacokinetic studies.     

High-performance liquid chromatography method for the quantification of pantoprazole in human plasma

A sensitive and selective HPLC method with UV detection (290 nm) was developed and validated for quantitation of pantoprazole, proton-pump inhibitor, in human plasma. Following a single-step liquid-liquid extraction with methyl tert-butyl ether/diethyl ether (70/30, v/v), the analyte and internal standard (zonisamide) were separated using an isocratic mobile phase of 10mM phosphate buffer (pH 6.0)/acetonitrile (61/39, v/v) on reverse phase Waters symmetry C18 column. The lower limit of quantitation was 20 ng/mL, with a relative standard deviation of less than 4%. A linear range of 20-5000 ng/mL was established. This HPLC method was validated with between-batch and within-batch precision of 1.3-3.2% and 0.7-3.3%, respectively. The between-batch and within-batch bias was -0.5 to 8.2 % and -2.5 to 12.1%, respectively. This validated method is sensitive and repeatable enough to be used in pharmacokinetic studies.     

High-performance liquid chromatography method for quantifying sphingomyelin in rat brain

A rapid, reproducible and accurate high-performance liquid chromatographic (HPLC) method for the quantitative determination of sphingomyelin in rat brain was developed and validated using normal-phase silica gel column, acetonitrile-methanol-water (65:18:17 (v/v)) at a flow rate of 1 ml/min, isocratic elution, UV detection at 207 nm and 1,2-dimyristoyl-sn-glycero-3-phosphocholine as an internal standard. Total run time was 10.0 min. The calibration curve was linear over the range of 0.025-0.4 mg/ml sphingomyelin (R2>0.99). The intra-day coefficient of variation ranged from 1.4% to 2.2%. The average inter-day coefficient of variation over a period of 4 days was 3.1%. The practical limit of detection was 0.005 mg/ml with a quantification limit of 0.01 mg/ml.