High-performance liquid chromatographic assay with UV detection for measurement of dihydrouracil/uracil ratio in plasma

A rapid, robust and sensitive HPLC method for analysis of uracil (U) and dihydrouracil (UH2) in plasma was developed using solid phase extraction and ultraviolet detection. Separation was achieved with a SymmetryShield RP18 column and an Atlantis dC18 column using a 10 mM potassium phosphate buffer as mobile phase. Compounds were eluted within 15 min without interference. Recovery was 80.4 and 80.6% for U and UH2. Calibration curves were linear from 2.5 to 80 ng/mL for U and 6.75 to 200 ng/mL for UH2. The LLQ was, respectively, 2.5 ng/mL for U, and 6.75 ng/mL for UH2. Within-run and between-run precision were less than 5.94% and inaccuracy did not exceed 7.80%. The overall procedure has been applied to correlate UH2/U ratio with dihydropyrimidine dehydrogenase activity in 165 cancer patients.     

Simple liquid chromatographic method for the determination of uracil and dihydrouracil plasma levels: a potential pretreatment predictor of 5-fluorouracil toxicity

5-Fluorouracil (5-FU) is a commonly used anti-cancer drug with notable activity in clinical practice, yet it causes significant unpredictable and often serious toxicity. Both 5-FU and uracil (U) are catabolised by dihydropyrimidine dehydrogenase (DPD) to form dihydrofluorouracil (FUH(2)) and dihydrouracil (UH(2)), respectively. A means of predicting toxicity before treatment would be more valuable. Variations in dihydropyrimidine dehydrogenase (DPD) activity between patients are at least partly responsible for variable toxicity. Measurement of the UH(2) to U ratio may be a measure of pyrimidine catabolism and thus be utilised to predict subsequent toxicity. We have developed an efficient extraction and detection method using HPLC for the simultaneous measurement of UH(2) and U in plasma. A single C(18) Spherisorb ODS2 (25 cm) column using isocratic elution was utilised. U, UH(2) and the internal standard 4-chlorouracil were detected at wavelengths of 257, 220, and 268 nm, respectively. The chromatographic run time was 45 min which is half that of other methods. The detection limit was 0.02 microM for U and 0.1 microM for UH(2) using only 0.5 ml of plasma for both compounds. The basal plasma concentrations of U and UH(2) in 23 individuals ranged from 0.025 to 0.27 microM and 0.4-1.7 microM, respectively. This simple method may permit the assessment of pyrimidine catabolism, and therefore allow prediction of the toxicities associated with the use of fluorinated pyrimidines.     

Validation of a liquid chromatographic method for the determination of ibuprofen in human plasma

A method based on LC-MS-MS is described for the determination of methyldopa in human plasma using dopa-phenyl-D3 as the internal standard. The method has a chromatographic run time of 5.5 min and was linear in the range of 20-5000 ng/ml. The limit of quantitation was 20 ng/ml, the intra-day precisions were 7.3, 5.4 and 4.3% and the intra-day accuracies were -8.0, -1.3 and -2.0% for 30, 600 and 3000 ng/ml, respectively. The inter-day precisions were 7.7, 0.5 and 0.7% and the inter-day accuracies were 0.2, -1.1 and -2.3%, respectively, for the above concentrations. This method was employed in a bioequivalence study of two tablet formulations of methyldopa.     

Measurement of endogenous uracil and dihydrouracil in plasma and urine of normal subjects by liquid chromatography-tandem mass spectrometry

A sensitive and specific HPLC-MS-MS method was developed for the determination of endogenous uracil (Ura) and its metabolite dihydrouracil (UH2) in human plasma and urine samples. Plasma samples were extracted with ethyl acetate-isopropanol (85:15, v/v) following added ammonium sulfate, and then separated on a Discovery Amide C16 column with 3% methanol solution as the mobile phase; urine samples were just centrifuged at 2500 g for detection. Quantitation was carried out by LC-MS-MS in the multiple reaction monitoring (MRM) mode. The limits of quantitation of the method for Ura and UH2 were 0.5 and 5 ng ml(-1) (for plasma), and 50 and 100 ng ml(-1) (for urine), respectively. This method can be useful to evaluate the activity of dihydropyrimidine dehydrogenase (DPD), a rate-limiting enzyme of the chemotherapy drug fluoropyrimidine, which will be helpful in investigating subject variation of DPD and adjusting clinical dosage in pyrimidine chemotherapy.     

Liquid chromatographic determination of penicillins by postcolumn alkaline degradation using a hollow-fiber membrane reactor

A high-performance liquid chromatographic method using a hollow-fiber membrane reactor is described for the determination of penicillins. This method involves separation of penicillins on a C18 column, postcolumn reaction with sodium hydroxide and mercury (II) chloride introduced into the main flow stream using sulfonated hollow-fiber membrane reactors immersed in each solution (4 M sodium hydroxide and 3 X 10(-2) M mercury (II) chloride plus 10(-2) M nitric acid), and detection at 290 nm based on the uv absorbance of the degradation products. At penicillin concentrations of 5 micrograms/ml, within- and between-run precisions (relative standard deviation) were 0.24-2.39 and 1.19-4.13%, respectively. The detection limits of the proposed method were 1-5 ng at a signal-to-noise ratio of 3. The method was applied to assays of ampicillin and its metabolites in human serum and urine.     

Determination of total iron in foods by isotope dilution fast atom bombardment mass spectrometry

A method employing isotope dilution fast atom bombardment mass spectrometry has been developed for the quantitative determination of iron in foods. Samples were spiked with an enriched source of 54Fe, digested with concentrated HNO3/H2SO4 and the iron extracted from 6 M HCl into diethyl ether. Bracketing procedures gave precisions ranging from 1.30-4.49% relative standard deviation (RSD) for isotope ratio measurements and 1.26-6.44% RSD for iron measurements.     

Application of a sensitive liquid chromatographic/tandem mass spectrometric method to a pharmacokinetic study of allylestrenol in healthy Chinese female volunteers

A sensitive, specific and rapid liquid chromatographic/tandem mass spectrometric (LC/MS/MS) assay for the determination of allylestrenol in human plasma was established. Plasma samples were extracted by tert-butyl ether and separated by LC/MS/MS using a Phenomenex Curosil-PFP column (250 mm x 4.6 mm ID, dp 5 microm) with a mobile phase of methanol-water (95:5, v/v). The analytes were monitored with atmospheric pressure chemical ionization (APCI) by selected reaction monitoring (SRM) mode. The linear calibration curves covered a concentration range of 0.04-20.0 ng/mL with lower limit of quantification (LLOQ) at 0.04 ng/mL. The mean extraction recovery of allylestrenol was greater than 81.8%. The intra- and inter-day precisions were less than 1.3% and 3.1% respectively, determined from quality control (QC) samples of three representative concentrations. The method has been successfully applied to determining the plasma concentration of allylestrenol and a clinical pharmacokinetics study in healthy Chinese female volunteers.     

Liquid chromatography with multi-channel electrochemical detection for the determination of epigallocatechin gallate in rat plasma utilizing an automated blood sampling device

A liquid chromatography method with multi-channel electrochemical detection was developed for the determination of epigallocatechin gallate (EGCG) in rat plasma. After administration of EGCG, blood samples were periodically collected by Culex (an automated blood sampling robot). EGCG was extracted from 50 μl of diluted blood (blood and saline at a ratio of 1:1) with ethyl acetate. Chromatographic separation was achieved within 10 min using a C₈ (150×4.6 mm) 5 μm column with a mobile phase containing 20 mM sodium monochloroacetate, pH 2.8 and 12% acetonitrile at a flow-rate of 1.2 ml/min. A four-channel detector with glassy carbon electrodes was used with applied potentials of +700, 600, 500, 400 mV vs. Ag/AgCl. The limit of detection was 2 ng/ml at a signal-to-noise ratio of 3:1 and the limit of quantitation was 5 ng/ml. The calibration curve was linear over the range of 5–800 ng/ml. The intra- and inter-assay precisions were in the range of 1.3–4.5% and 2.2–4.4%, respectively. Using this method it was possible to determine plasma concentration following a single dose of EGCG to rats with good accuracy and precision. Thus the pharmacokinetic properties of EGCG in rats can be examined for intravenous, intraperitoneal and oral dosing.     

Liquid chromatographic assay with fluorescence detection to determine ajmaline in serum from patients with suspected Brugada syndrome

Ajmaline is a sodium channel blocking, class 1A anti-arrhythmic drug. It has gained renewed interest in the field of cardiology as a diagnostic agent to reveal the electrocardiographic characteristics in patients with suspected Brugada syndrome. We developed a simple and precise high-performance liquid chromatographic assay to determine ajmaline in serum of patients. The samples were pre-treated using protein precipitation with perchloric acid and the extract was injected into the chromatographic system. The system consisted of an end-capped octadecyl silica column with isocratic elution using perchloric acid in a water-acetonitrile mixture. Ajmaline was detected by fluorescence at 290 and 355 nm for excitation and emission, respectively. The assay was validated in a 21-5300 ng/ml concentration range, the lower limit of quantification was 25 ng/ml. Within day precisions were 1.3-3.9%, between day precisions 2-7% and accuracies were between 95 and 99% for the whole calibration range. The drug was shown to be chemically stable under all relevant conditions. This assay has been successfully applied to pharmacokinetic-pharmacodynamic evaluations of intravenous ajmaline administration to patients with suspected Brugada syndrome.     

A HPLC method for determination of nicousamide in dog plasma and its application to pharmacokinetic studies

A sensitive and reproducible high performance liquid chromatography (HPLC)-UV method for determination of nicousamide, an inhibitor of rennin and transforming growth factor-beta1 (TGF-beta1) type II receptors, has been developed and validated. Following acetonitrile deproteiniation, samples were separated by isocratic reversed-phase HPLC on an Aichrom Bond-AQ C(18) column and quantified using UV detection at 320 nm. The mobile phase was acetonitrile/water (ratio 62:38 containing 0.1% H(3)PO(4)), with a flow-rate of 1.0 ml/min. A linear curve over the concentration range 5-200 ng/ml (r(2)=0.9978) was obtained. The coefficients of the variation for the intra- and inter-day precisions ranged from 1.4-10.7% and 1.8-7.1%, respectively. The percentage of relative recovery was 91.56-105.45%. The method was used to determine the plasma concentration-time profiles for nicousamide after oral doses of 30, 100 and 300 mg/kg in dogs. A nonlinear pharmacokinetics was found in dogs at doses from 30 to 300 mg/kg. Following 30 mg/kg oral dose, the C(max) and AUC in females were lower than that in male. There is a potential for accumulation in dogs following multiple doses.     

Salmonella typhi Ty2 OmpC Porin Induces Bactericidal Activity on U937 Monocytes

The immunogenic effect of Salmonella typhi OmpC porin during typhoid fever in humans was evaluated in vitro. Peripheral blood mononuclear cells from 17 patients were challenged with outer membrane preparations from Escherichia coli UH302 and UH302/pSTP2K2 strains, both lacking E. coli OmpF and OmpC porins, although UH302/pSTP2K2 expressed a plasmid-encoded S. typhi Ty2 OmpC. The mononuclear cell supernatants, immunized in vitro with OmpC antigen, derived from 10 out of 17 patients activated U937 bactericidal capacity. In contrast, the supernatants from the immunization with outer membrane preparation lacking S. typhi Ty2 OmpC induced a significantly reduced bactericidal capacity of U937 cells. This procedure should prove useful for in vitro characterization of cellular immunogens from exclusive human pathogens.     

Quantification of puerarin in plasma by on-line solid-phase extraction column switching liquid chromatography-tandem mass spectrometry and its applications to a pharmacokinetic study

A highly precise, automatic and rapid method for quantification of puerarin in canine and human plasma using an on-line solid-phase extraction (SPE) column switching procedure combined with liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS) was developed. The eluent of SPE column consisted of acetonitrile/methanol/0.1% formic acid (25/25/50) at a flow rate of 0.2mLmin(-1). Puerarin was analyzed by a linear ion trap mass spectrometer, LTQ-MS, operating in the negative ion and selective reaction monitoring (SRM) acquisition mode. Method validation results demonstrated that the linear calibration curve covered a wide range of 0.39-400.00ngmL(-1), the correlation coefficients (r(2)) were above 0.999. The lower limit of detection (LLOD) with the signal-to-noise (S/N) ratio higher than 12 was 0.39ngmL(-1). The intra- and inter-batch precisions were less than 7.61% and 6.42%, respectively. The accuracy was well within the accept limit. The on-line SPE column switching HPLC-MS system was applied to pharmacokinetic (PK) study of puerarin after a single orally dose in beagles. And the optimum conditions were successfully utilized to quantify puerarin in human plasma, which indicated the feasibility and the reliability of this method for application in preclinical and clinical PK studies of isoflavone drugs.     

Effects of hypothermia on rat brain pHi and phosphate metabolite regulation by 31P-NMR

The effects of arterial alphastat regulation on brain intracellular pH (pHi) and several phosphate metabolites were assessed in anesthetized rats during hypothermia (28.6 +/- 0.2 degrees C) and normothermia (36.2 +/- 0.2 degrees C) by using 31P high-field (8.5 T) nuclear magnetic resonance (NMR). There were significant differences in pHi and metabolite ratios at the two temperatures under conditions of equal minute ventilation. During hypothermia, the brain pHi was 0.09 U higher, the phosphocreatine-to-inorganic phosphate (PCR/Pi) ratio 49% larger, and Pi-to-ATP 20% lower than at normothermia. These changes were fully reversible on warming the animal. The change in brain pHi/temperature was -0.011U/degrees C (95% confidence interval -0.007 to -0.016). The brain's ability to regulate its pHi and phosphate metabolism during hypercapnic acid-base stress was studied by using 10% CO2 ventilation. Hypothermic rats showed a larger fall in brain pHi (0.145 +/- 0.01 U, 7.15-7.01) with 10% CO2 than normothermic rats (0.10 +/- 0.02 U, 7.06-6.96). Similarly ventilated rats had a larger fall in arterial pH with 10% CO2 at hypothermia (0.36 +/- 0.04 U) than normothermia (0.24 +/- 0.01 U), so the delta brain pH/delta arterial pH was the same at both temperatures. The brain PCr-to-Pi ratio decreased approximately 20% during 10% CO2 breathing in both hypothermic and normothermic animals. Brain pHi and metabolite ratios returned to base line 30-50 min after CO2 washout in both groups. In summary, lowering body temperature while maintaining constant ventilation leads to changes in brain pHi and metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anthurium roots for micropropagation and Agrobacterium tumefaciens-mediated gene transfer

Growth of Blechnum spicant gametophytes was optimal in MS liquid medium, a 16-h photoperiod, and it was unaffected by variation of the pH between 4.7 and 8.7. Antheridia were observed during all developmental stages of the gametophyte: filamentous, spatulate or cordate and their formation was induced by compounds excreted into the culture medium by mature gametophytes. This antheridiogen activity was found in the fractions corresponding to free and apolar esters of gibberellins. IBA at 5 μM and 50 μM, and BA at 50 μM inhibited antheridiogen. Exogenous application of GA₃ allowed spore germination but strongly inhibited gametophyte development; the two dimensional state was not reached. This revised version was published online in June 2006 with corrections to the Cover Date.     

Matrix solid-phase dispersion extraction coupled with HPLC-diode array detection method for the analysis of sesquiterpene lactones in root of Saussurea lappa C.B.Clarke

We developed a reliable and effective method to determine costunolide and dehydrocostuslactone in the root of Saussurea lappa C. B.Clarke using matrix solid-phase dispersion (MSPD) extraction, HPLC separation and diode array detection (DAD). Several extraction parameters for the MSPD were optimized. Florisil was chosen as dispersing adsorbent with methanol as elution solvent. The ratio of Florisil to sample was selected to be 4:1 and no additional clean-up steps were needed. Linearities (r>0.9995) were determined to be in the range of 22.5-360.0 μg/mL for costunolide and 25.0-400.0 μg/mL for dehydrocostuslactone. Intra- and inter-day precisions were also determined with a relative standard deviation (RSD) less than 3.2%. The limits of detection were found to be 0.122 μg/mL for costunolide and 0.135 μg/mL for dehydrocostuslactone. The recoveries were in the range of 92.5-99.8% with relative standard deviations ranged from 1.2% to 3.5%. The proposed MSPD method required shorter time and lower solvent volume than maceration-ultrasonic and Soxhlet extraction methods.     

Cellobiase from Termitomyces clypeatus: activity and secretion in presence of glycosylation inhibitors

In presence of the glycosylation inhibitors, 2-deoxy-d-glucose (1 mg/ml), tunicamycin (30 μg/ml), 1-deoxynojirimycin (30 μg/ml) and d-glucono-δ-lactone (1 mg/ml), total cellobiase activity, in the extracellular, intracellular and cell bound fractions, of the fungus Termitomyces clypeatus grown in 20 ml cellobiose medium (1%, w/v) increased by 50-, 1.8-, 2.4-, 1.3-fold, respectively, with respect to control medium (16.3 U). The inhibitors also stimulated secretion of 95% of the total protein in culture medium, except d-glucono-δ-lactone which released 60% of the total protein. 2-Deoxy-d-glucose (1 mg/ml) led to production of extracellular cellobiase up to 40 U/ml, whereas in absence of the inhibitors only 0.59 U/ml enzyme was detected.     

Nuclear magnetic resonance spectroscopy and molecular modeling reveal that different hydrogen bonding patterns are possible for G.U pairs: one hydrogen bond for each G.U pair in r(GGCGUGCC)(2) and two for each G.U pair in r(GAGUGCUC)(2)

G.U pairs occur frequently and have many important biological functions. The stability of symmetric tandem G.U motifs depends both on the adjacent Watson-Crick base pairs, e.g., 5'G > 5'C, and the sequence of the G.U pairs, i.e., 5'-UG-3' > 5'-GU-3', where an underline represents a nucleotide in a G.U pair [Wu, M., McDowell, J. A., and Turner, D. H. (1995) Biochemistry 34, 3204-3211]. In particular, at 37 degrees C, the motif 5'-CGUG-3' is less stable by approximately 3 kcal/mol compared with other symmetric tandem G.U motifs with G-C as adjacent pairs: 5'-GGUC-3', 5'-GUGC-3', and 5'-CUGG-3'. The solution structures of r(GAGUGCUC)(2) and r(GGCGUGCC)(2) duplexes have been determined by NMR and restrained simulated annealing. The global geometry of both duplexes is close to A-form, with some distortions localized in the tandem G.U pair region. The striking discovery is that in r(GGCGUGCC)(2) each G.U pair apparently has only one hydrogen bond instead of the two expected for a canonical wobble pair. In the one-hydrogen-bond model, the distance between GO6 and UH3 is too far to form a hydrogen bond. In addition, the temperature dependence of the imino proton resonances is also consistent with the different number of hydrogen bonds in the G.U pair. To test the NMR models, U or G in various G.U pairs were individually replaced by N3-methyluridine or isoguanosine, respectively, thus eliminating the possibility of hydrogen bonding between GO6 and UH3. The results of thermal melting studies on duplexes with these substitutions support the NMR models.     

Maternal cigarette smoking, Down syndrome in live births, and infant race.

Previous studies have suggested that maternal smoking is negatively associated with a Down syndrome live birth. We analyzed the data of the U.S. Perinatal Collaborative Study in a search for racial variation in Down syndrome risk factors. There were 22 cases in 25,346 live births to smoking mothers (4/10,780 blacks, 18/13,320 whites, and 0/1,246 other races) and 42/29,130 live births to nonsmoking mothers (24/14,665 blacks, 14/11,694 whites, and 4/2,771 others). The crude overall rates per 1,000 live births were 0.4 in black smokers and 1.6 in black nonsmokers but 1.4 in white smokers and 1.2 in white non-smokers. Adjusted for maternal age, the summary relative risk for a Down syndrome live birth to a smoking mother was 0.2 in blacks (95% interval 0.1-0.7) but 1.2 in whites (95% interval 0.6-2.5). Stratification on variables associated with socioeconomic status or gestational age at time of entry into the study did not alter the racial difference. A comparison of smokers with those who never smoked revealed essentially the same trends. Among all nonsmokers the ratio of the maternal age-adjusted risks for a Down syndrome live birth in whites compared with blacks was 0.7 (95% interval 0.3-1.3), and among all smokers this ratio was 3.6 (95% interval 1.3-9.9). If the results are not attributable to statistical fluctuation or undetected confounding, then differences in the probability of intrauterine survival of the Down syndrome fetus would appear to be one plausible explanation for the difference.     

Determination of lactate dehydrogenase in human erythrocytes by capillary electrophoresis with electrochemical detection

Capillary electrophoresis (CE) was employed to analyze lactate dehydrogenase (LDH) in human erythrocytes using an amperometric detector with a carbon fiber micro-disk bundle electrode. LDH activity was measured by determining the amount of NADH generated by LDH through a enzyme-catalyzed reaction between NAD(+) and lithium lactate. The factors influencing the enzyme-catalyzed reaction, separation and detection were examined and optimized. The following conditions were suitable for the determination of LDH: running buffer, 5.0 x 10(-2)mol/l Tris-HCl (pH 7.5); separation voltage, 20.0 kV; detection potential, 1.00 V (versus saturated calomel electrode (SCE)). The conditions of enzyme-catalyzed reaction were: reaction buffer, 5.0 x 10(-2)mol/l Tris-HCl (pH 9.3); substrates, 5.0 x 10(-2)mol/l lithium lactate and 5.0 x 10(-3)mol/l NAD(+); reaction time, 10 min. The concentration limit of detection (LOD) of the method was 0.017 U/ml at a signal-to-noise (S/N) ratio of 3, which corresponded to 1.10 x 10(-10)mol/l, and the mass LOD was 2 x 10(-20)mol. The linear dynamic range was 0.039-4.65 U/ml for the injection voltage of 5.0 kV and injection time of 10s. The relative standard deviation (R.S.D.) was 0.85% for the migration time and 1.8% for the electrophoretic peak area. The method was applied to determine LDH in human erythrocytes. The recovery of the method was between 98 and 101%.     

超声激活血卟啉对S180 肿瘤细胞表面EGFR 表达量的影响

目的：探讨超声激活血卟啉处理S180肿瘤细胞后膜表面EGFR表达量的变化。方法：将腹水瘤细胞随机分为四组,U组和UH组细胞分别于频率1．8MHz、2．0W／cm^2的超声装置中照射3min,并分别在1h3h5h后取材,应用免疫组化方法在光镜下观察EGFR的表达情况。结果：1h、3h取材,U组和UH组平均光密度值明显低于Cr组和H组,UH组最低。而5h取材时,UH组平均光密度值显著下降,其它组基本无变化。结论：提示在高频低强度处理下,随着时间的延长,超声激活HpD对EGFR的抑制作用增加,显示可能是基因调控使EGFR表达下调,从而使肿瘤细胞增殖减慢。