Plasma estrogen suppression with aromatase inhibitors evaluated by a novel, sensitive assay for estrone sulphate

Aromatase inhibition is a well-defined treatment option for postmenopausal breast cancer. Although several aromatase inhibitors such as aminoglutethimide, formestane, fadrozole have been found to inhibit in vivo aromatization by>85%, previous studies reported plasma estrogen levels to be sustained at approximately 20–50% of their control level during treatment with these drugs. The discrepancy could be due to lack of sensitivity or non-specific crossreactions in the radioimmunoassay (RIA) methods. Mean plasma levels of estrone (E₁) and estradiol (E₂) in postmenopausal women are approximately 80 and 20 pmol/l, respectively; on the contrary, mean plasma levels of the estrogen conjugate estrone sulphate (E₁S) are approximately 4–500 pmol/l. Most RIA methods for plasma E₂ and E₁ measurements have sensitivity limits in the range of 2–3 and 7–10 pmol/l, respectively; accordingly, the suppression of plasma estrogens by more than 80–90% will produce hormone values below the sensitivity limit of the method in many patients. Recently, we developed a new method to determine plasma E₁S. This assay has a sensitivity limit of 2.7 pmol/l. In theory, this method may allow the determination of plasma E₁S levels suppressed to less than 2% of control values in the majority of patients. Using this method, we found different aromatase inhibitors such as formestane, aminoglutethimide, formestane and aminoglutethimide administered in concert or anastrozole to suppress plasma E₁S levels down to 24, 13, 7 and 4%, respectively. The suppression of plasma E₁S evaluated with this method thus approaches the percentage aromatase inhibition measured with tracer studies.     

High-performance liquid chromatographic analysis of dipyridamole in plasma and whole blood

A rapid, sensitive, and specific high-performance liquid chromatographic method is described for the quantitative analysis of dipyridamole in plasma and whole blood. The method involves a single extraction of an alkalinized sample with diethyl ether followed by evaporation of the organic solvent and ion-pair chromatography using fluorescence detection. The lower limit of sensitivity for dipyridamole is 1 ng/ml. Concentrations of dipyridamole between 1 and 500 ng per sample are measured with an average coefficient of variation of 4.5% in plasma and 7.4% in whole blood.     

Effect of in vitro cultivation on the stability of resistance of Trypanosoma brucei brucei to diminazene, isometamidium, quinapyramine, and Mel B

A Trypanosoma brucei brucei stock resistant to diminazene aceturate, isometamidium chloride, quinapyramine sulfate, and Mel B was grown in vitro and its response to these drugs compared to that of a drug-sensitive trypanosome stock. There was little if any change of drug sensitivity after in vitro propagation as bloodstream forms for 120, 177, and 275 days and after in vitro transformation of bloodstream forms into procyclic, epimastigote, and finally metacyclic forms. Drug resistance was stable during in vitro maintenance in the absence of drugs in both culture systems. The response of resistant and sensitive T. b. brucei to diminazene in vitro correlated with their sensitivity pattern in vivo. Thus, in vitro techniques can be used to study drug resistance in trypanosomes.     

Ferrocene-based Diels-Alder derivatization for the determination of 1alpha-hydroxyvitamin D3 in rat plasma by high-performance liquid chromatography-electrospray tandem mass spectrometry

A specific and sensitive liquid chromatography-electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method has been developed for the determination of 1alpha-hydroxyvitamin D(3) (1alphaOHD(3)) in rat plasma. A new ferrocene-based Cookson-type reagent, 4-ferrocenylmethyl-1,2,4-triazoline-3,5-dione (FMTAD), designed and synthesized to be highly sensitive to vitamin D analogs in ESI, considerably improved the detection limit with 250 fg (359 amol)/injection. 1alphaOHD(3) in rat plasma was extracted with acetonitrile and then purified using Oasis HLB 96-well plates. After the precolumn derivatization with FMTAD, samples were subjected to LC/ESI-MS/MS employing a column-switching system. This method achieved a lower limit of quantitation of 5 pg from 0.1-mL plasma aliquots and 200-fold sensitivity of that without derivatization. The calibration curve (0.05-15 ng/mL) exhibited acceptable linearity (r>0.9966), intraassay precision ranged from 3.8 to 9.6%, interassay precision ranged from 3.0 to 17.0%, and accuracy was within 81.4-112.0%. This FMTAD derivatization method is considered very useful for determination of vitamin D analogs in ESI and applicable for biological samples.     

The effects of diminazene aceturate and ceftriaxone on ram sperm

Effects of diminazene aceturate and ceftriaxone disodium were evaluated on sperm quality of rams. Daily intramuscular injections of diminazene (6 mg/kg) or ceftriaxone (28.5 mg/kg) were given to each of seven Akkaraman rams assigned per drug for two days. Semen samples were collected from the rams at post-treatment 1, 4, 24, 48, 72, 144, 288 and 336 h and examined for sperm characteristics and hyaluronidase activity. Results showed that use of ceftriaxone and diminazene caused significant (P<0.01) decreases in sperm concentration, volume and motility compared to control group within 288 h post-treatment. In addition, hyaluronidase activity increased significantly (P<0.01) in semen of rams treated with ceftriaxone while remained unchanged in those received diminazene. In conclusion, diminazene aceturate and ceftriaxone disodium did not have any deleterious effect on hyaluronidase enzyme. However, both drugs caused impairment of sperm in rams during the 288 h.     

Liquid chromatography-tandem mass spectroscopy assay for quercetin and conjugated quercetin metabolites in human plasma and urine

A sensitive and specific method was developed and validated for the quantitation of quercetin in human plasma and urine. The application of liquid chromatography-tandem mass spectrometry (LC/MS/MS) with a TurboIonspray (TIS) interface in negative mode under multiple reactions monitoring was investigated. Chromatographic separation was achieved on a C12 column using a mobile phase of acetonitrile/water with 0.2% formic acid (pH 2.4) (40/60, v/v). The detection limit was 100 pg/ml and the lower limit of quantification was 500 pg/ml for plasma samples; the detection limit was 500 pg/ml and the lower limit of quantification was 1 ng/ml for urine samples. The calibration curve was linear from 1 to 800 ng/ml for plasma samples and was linear from 1 to 200 and 50 to 2000 ng/ml for urine samples. All the intra- and inter-day coefficients of variation were less than 11% and intra- and inter-day accuracies were within +/-15% of the known concentrations. This represents a LC/MS/MS assay with the sensitivity and specificity necessary to determine quercetin in human plasma and urine. This assay was used to determine both parent quercetin and the quercetin after enzymatic hydrolysis with beta-glucuronidase/sulfatase in human plasma and urine samples following the ingestion of quercetin 500 mg capsules.     

Simple GC-FID method development and validation for determination of alpha-tocopherol (vitamin E) in human plasma

This paper describes the development and validation of a novel GC-FID method for the determination of alpha-tocopherol concentration in human plasma which does not requires derivatization. The standard solutions and the plasma working solutions were prepared in absolute ethanol. To determine the concentration of alpha-tocopherol in human plasma, an aliquot of the plasma sample was deproteinized with ethanol. alpha-tocopherol was extracted with a mixture of hexane and dichloromethane (9:1). GC separation was performed using a HP-5 capillary column. Nitrogen was used as carrier gas at a flow-rate of 2 ml min(-1). Calibration curves were linear over the concentration range 1-30 microg ml(-1) (for standard solutions and solutions without endogenous alpha-tocopherol in plasma) and 5-34 microg ml(-1) (for solutions with endogenous alpha-tocopherol in plasma). Absolute recovery, precision, sensitivity and accuracy assays were carried out. The analytical recovery of alpha-tocopherol from plasma averaged 97.44%. The limit of quantification (LOQ) and the limit of detection (LOD) of method for standard samples were 0.35 microg.ml(-1) and 0.30 microg.ml(-1), respectively. Within-day and between-day precision, expressed as the relative standard deviation (RSD) were less than 4%, and accuracy (relative error) was better than 8%. This novel method, developed and validated in our laboratory, could be successfully applied to the in-vivo determination of alpha-tocopherol. The endogenous alpha-tocopherol amounts in blood of twelve healthy volunteers with no vitamin drug usage were measured with this method.     

Analysis of betamethasone in rat plasma using automated solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry. Determination of plasma concentrations in rat following oral and intravenous administration

A method is described for the determination of betamethasone in rat plasma by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The analyte was recovered from plasma by solid-phase extraction and subsequently analyzed by LC-MS-MS. A Packard Multiprobe II, an automated liquid handling system, was employed for the preparation and extraction of a 96-well plate containing unknown plasma samples, standards and quality control samples in an automated fashion. Prednisolone, a structurally related steroid, was used as an internal standard. Using the described approach, a limit of quantitation of 2 ng/ml was achieved with a 50 microl aliquot of rat plasma. The described level of sensitivity allowed the determination of betamethasone concentrations and subsequent measurement of kinetic parameters of betamethasone in rat. Combination of automated plasma extraction and the sensitivity and selectivity of LC-MS-MS offers a valuable alternative to the methodologies currently used for the quantitation of steroids in biological fluids.     

Sensitive method for the quantitative determination of proguanil and its metabolites in rat blood and plasma by liquid chromatography-mass spectrometry

A sensitive, simple and fast liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for the determination of proguanil (PG) and its metabolites, cycloguanil (CG) and 1-(4-chlorophenyl)biguanide (4CPB), was developed and validated over a concentration range of 1-2000 ng/mL using only 50 microL of blood or plasma. After a simple solvent precipitation procedure, the supernatant was analysed directly by HPLC-MS/MS. Separation was achieved using an ethyl-linked phenyl reverse phase column with polar endcapping with an acetonitrile-water-formic acid gradient. Mass spectrometry was performed using a triple quadrupole mass spectrometer operating in positive electrospray ionization mode. The elution of PG (254.07-->169.99), CG (252.12-->195.02) and 4CPB (212.06-->153.06) was monitored using selected reaction monitoring. The three compounds and the internal standard (chloroproguanil) were well separated by HPLC and no interfering peaks were detected at the usual concentrations found in blood and plasma. The limit of quantification of PG and CG was 1 ng/mL and 5 ng/mL for 4CPB in rat blood and plasma. The extraction efficiency of PG, CG and 4CPB from rat blood and plasma was higher than 73%. The intra- and inter-assay variability of PG, CG and 4CPB were within 12% and the accuracy within +/-5%. This new assay offers higher sensitivity and a much shorter run time over earlier methods.     

Chromatographic and immunochemical approaches to the analysis of the HIV protease inhibitor saquinavir in plasma

The development of the HIV protease inhibitor saquinavir (Ro 31-8959) required a range of analytical methods for its measurement in biological fluids. This paper describes the development of isocratic, reverse-phase HPLC/UV methods for the routine measurement of plasma levels of the drug together with a more sensitive radioimmunoassay. The performance of the two assays is compared with that of an HPLC/MS/MS method previously published and has been shown to be satisfactory, with coefficients of variation of calibration standards and quality control samples within the usual outside limits of +/- 15%. The HPLC/UV method can be routinely applied for concentrations down to 10-20 ng/ml and a lower limit of quantification of 1 ng/ml from 1 ml of human plasma is possible. The radioimmunoassay was developed for the specific measurement of saquinavir concentrations in human, HIV-positive plasma samples and has a lower limit of quantification of 0.5-1.0 ng/ml. Some preliminary findings suggested that it might not be specific in rat plasma and no attempts have been made to quantify any nonclinical samples with this technique. If still greater sensitivity is required, recourse can be made to the HPLC/MS/MS assay.     

Detection, quantification and confirmation of anabolic steroids in equine plasma by liquid chromatography and tandem mass spectrometry

Anabolic androgenic steroids are related to the male sex hormones and are abused in equine sports. In an effort to deter the abuse of anabolic steroids, a sensitive LC-MS/MS method was developed for detection, quantification and confirmation of eight major anabolic steroids (testosterone, normethandrolone, nandrolone, boldenone, methandrostenolone, tetrahydrogestrinone (THG), trenbolone, and stanozolol) in equine plasma. Formation of solvent adduct ions of the analytes was observed under electrospray ionization (ESI) conditions, and desolvation of the solvent adduct ions by source collision-induced decomposition (CID) increased the abundance of the [M+H]+ ions as well as the multiple-reaction monitoring (MRM) signals. ESI (+) and APCI (+) were compared with respect to sensitivity for the analytes and the former provided better sensitivity. The matrix effect on ion suppression or enhancement was evaluated, and was negligible. Confirmation of the analytes was performed using criteria of three ion transitions and LC retention time of each analyte. The limit of detection (LOD) and quantification (LOQ) was 25 pg/mL. The limit of confirmation (LOC) was 25 pg/mL for boldenone; 50 pg/mL for normethandrolone, nandrolone, and methandrostenolone; and 100 pg/mL for testosterone, THG, trenbolone, and stanozolol. The analytes were evaluated for stability and found to be stable in plasma for 24h at room temperature, 13 days at 4 degrees C, and 34 days at -20 and -70 degrees C. The method was successfully applied to analyses of equine plasma samples for pharmacokinetics study. This method is sensitive and useful for detection, quantification and confirmation of these anabolic steroids in equine plasma.     

Superparamagnetic poly(methyl methacrylate) beads for nattokinase purification from fermentation broth

An effective method for purification of nattokinase from fermentation broth using magnetic poly(methyl methacrylate) (PMMA) beads immobilized with p-aminobenzamidine was proposed in this study. Firstly, magnetic PMMA beads with a narrow size distribution were prepared by spraying suspension polymerization. Then, they were highly functionalized via transesterification reaction with polyethylene glycol. The surface hydroxyl-modified magnetic beads obtained were further modified with chloroethylamine to transfer the surface amino-modified magnetic functional beads. The morphology and surface functionality of the magnetic beads were examined by scanning electron microscopy and Fourier transform infrared. An affinity ligand, p-aminobenzamidine was covalently immobilized to the amino-modified magnetic beads by the glutaraldehyde method for nattokinase purification directly from the fermentation broth. The purification factor and the recovery of the enzyme activity were found to be 8.7 and 85%, respectively. The purification of nattokinase from fermentation broth by magnetic beads only took 40 min, which shows a very fast purification of nattokinase compared to traditional purification methods.     

Determination of the 4-monohydroxy metabolites of perhexiline in human plasma, urine and liver microsomes by liquid chromatography

The use of perhexiline (PHX) is limited by hepatic and neurological toxicity associated with elevated concentrations in plasma that are the result of polymorphism of the cytochrome P450 2D6 isoform (CYP2D6). PHX is cleared by hepatic oxidation that produces three 4-monohydroxy metabolites: cis-OH-PHX, trans1-OH-PHX and trans2-OH-PHX. The current study describes an HPLC-fluorescent method utilising pre-column derivatization with dansyl chloride. Following derivatization, the metabolites were resolved on a C18 column with a gradient elution using a mobile phase composed of methanol and water. The method described is suitable for the quantification of the metabolites in human plasma and urine following clinical doses and for kinetic studies using human liver microsomes. The method demonstrates sufficient sensitivity, accuracy and precision between 5.0 and 0.01, 50.0 and 0.2 and 1.0 and 0.005 mg/l in human plasma, urine and liver microsomes, respectively, with intra-assay coefficients of variation and bias <15%, except at the lowest limit of quantification (<20%). The inter-assay coefficients of variation and bias were <15%. The application of this method to plasma and urine samples of five CYP2D6 extensive metaboliser (EM) patients at steady state with respect to PHX dosing determined that the mean (+/-S.D.) renal clearances of trans1-OH-PHX and cis-OH-PHX were 1.58+/-0.35 and 0.16+/-0.06l/h, respectively. The mean (+/-S.D.) dose recovered in urine as free and glucuronidated 4-monohydroxy PHX metabolites was 20.6+/-11.6%.     

Diminazene aceturate associated with sodium selenite and vitamin E in the treatment of Trypanosoma evansi infection in rats

The aim of this study was to evaluate the utilization of a standard treatment with diminazene aceturate against the infection caused by Trypanosoma evansi, associated to sodium selenite and vitamin E. In vitro tests showed trypanocidal effect related to the treatment with diminazene aceturate and sodium selenite, but vitamin E had no harmful effect on the trypanosomes. In vivo experiments utilized a total of 72 adult outbreed females rats, separated into 9 groups (A, B, C, D, E, F, G, H and I), 8 animals each. Group A was the uninfected group; groups B to I were infected with 0.2 mL of blood containing 10⁶ trypanosomes. Parasitemia was estimated daily by microscopic examination of blood smears. Group B served as positive control; group C was treated with diminazene aceturate; group D with sodium selenite; group E with vitamin E; group F received an association of diminazene aceturate and sodium selenite; group G received an association of diminazene aceturate and vitamin E; group H received an association of diminazene aceturate, sodium selenite and vitamin E, and group I received an association of sodium selenite and vitamin E. Diminazene aceturate was administrated in a single dose on the 3rd day post infection (PI). Sodium selenite and vitamin E were administered at the 3rd and 23rd day PI. In vivo tests showed increase of longevity in groups treated with diminazene aceturate associated with sodium selenite (groups F and H). No difference was found between groups C and E, thus the vitamin E did not increase the efficacy of treatment against T. evansi when associated to diminazene aceturate. The curative efficacy of treatments was 37.5, 87.7, 37.7 and 75% to the groups C, F, G and H, respectively. Other treatments showed no efficacy. The sodium selenite when combined with chemotherapy may represent an alternative in the treatment of trypanosomosis.     

High performance liquid chromatographic determination of N-butyryl glucosamine in rat plasma

PURPOSE: A high performance liquid chromatography (HPLC) method for determination in plasma of N-butyryl glucosamine (GLBU), a highly water-soluble compound with no chromophore was developed. METHOD: To 100 muL of plasma containing GLBU was added fucose as internal standard. GLBU and fucose were derivatized using 1-phenyl-3-methyl-5-pyrazolone in the presence of sodium hydroxide at 70 degrees C for 30 min. The solution was neutralized with hydrochloric acid and the excess derivatizing reagent was extracted with chloroform. The aqueous layer was injected into an isocratic HPLC system consisting of an autoinjector, a single pump and a UV detector set at 245 nm. Two different 25 cm reversed phase columns were used, a 4 and a 10 microm C(18) columns. The mobile phase was a mixture of phosphate buffer (pH 7) and acetonitrile (80:20), which was run through a pump at a flow rate of 1.0 mL/min at ambient temperature. RESULTS: Derivatized fucose and GLBU appeared 24 and 28 min, and at 34 and 37 min using 4 and 10 microm columns, respectively. The assay was linear over the range of 0.2-200 microg/mL with a limit of quantification of 0.2 and 1 microg/mL for the 4 and 10 microm columns, respectively. The method was applied to the determination of GLBU in rat plasma after oral administration of 233 mg/kg of GLBU. CONCLUSION: The present assay is precise, and accurate with sufficient sensitivity for pharmacokinetic studies following therapeutically relevant doses.     

Determination of a new blood glucose-lowering agent in biological fluids by capillary gas chromatography using electron-capture detection

A gas chromatographic method for the determination of 5-(4-chlorophenyl)-2-[(4-methylphenyl)sulphonyl]-4-pentynoic acid (I) in plasma (serum) and urine has been developed. After an extraction process, the cleaned-up organic extract was derivatized with diazomethane at ambient temperature. Results are evaluated from peak-height ratios with respect to the appropriate internal standard. The detection limit following extraction of a 1-ml plasma sample is about 20 ng/ml.     

Reversible inhibition of cell multiplication in vitro by inhibitors of arginine esteroproteases

Two classes of active-site specific inhibitors of trypsin-like proteases have been shown to inhibit reversibly the multiplication of eukaryotic cells in vitro. The competitive inhibitors p-aminobenzamidine and benzamidine were found to arrest the growth of normal and transformed mouse fibroblasts and human KB cells. The inhibition of cell multiplication occurred within 24 h and was accompanied by substantial decreases in the rates of DNA and protein synthesis. The rate of RNA synthesis was relatively unaffected by the protease inhibitors. In agreement with the known inhibition constants (K_i) for their action against trypsin, p-aminobenzamidine was a much more effective inhibitor of cell multiplication than benzamidine. In addition, tosyllysine chloromethyl ketone (Tos-LysCH₂Cl), an active-site titrant and irreversible inhibitor of trypsin, was found to cause a reversible inhibition of growth. These results suggest that an essential protease activity is necessary for cell multiplication. However, in the case of mouse L-cells, all of the inhibitors and particulary p-aminobenzamidine caused excessive accumulation of lactate in the extracellular medium. This observation, which suggests the possibility of additional sites of action of these compounds in cells, was found to depend upon the cell type and appears to be unrelated to the inhibition of growth.     

Efficacy study of novel diamidine compounds in a Trypanosoma evansi goat model

Three diamidines (DB 75, DB 867 and DB 1192) were selected and their ability to cure T. evansi experimentally infected goats was investigated. A toxicity assessment and pharmacokinetic analysis of these compounds were additionally carried out. Goats demonstrated no signs of acute toxicity, when treated with four doses of 1 mg/kg/day (total dose 4 mg/kg). Complete curative efficacy of experimentally infected goats was seen in the positive control group treated with diminazene at 5 mg/kg and in the DB 75 and DB 867 groups treated at 2.5 mg/kg. Drug treatment was administered once every second day for a total of seven days. Complete cure was also seen in the group of goats treated with DB 75 at 1.25 mg/kg. DB 1192 was incapable of curing goats at either four-times 2.5 mg/kg or 1.25 mg/kg. Pharmacokinetic analysis clearly demonstrated that the treatment failures of DB 1192 were due to sub-therapeutic compound levels in goat plasma, whilst compound levels for DB 75 and DB 867 remained well within the therapeutic window. In conclusion, two diamidine compounds (DB 75 and DB 867) presented comparable efficacy at lower doses than the standard drug diminazene and could be considered as potential clinical candidates against T. evansi infection.     

Determination of nalidixic acid and its two major metabolites in human plasma and urine by reversed-phase high-performance liquid chromatography

This paper describes a precise and sensitive method for analysis of nalidixic acid and its two major metabolites in plasma and urine following the oral administration of a therapeutic dose in humans. After addition of an internal standard (oxolinic acid), 1-ml samples of plasma or urine are extracted at acidic pH with chloroform. The extracts are purified by re-extraction with sodium hydroxide solution and then chloroform. The final extracts are evaporated to dryness, reconstituted in mobile phase and injected into a high-performance liquid chromatograph equipped with RP-8 column and UV detector operating at 254 nm. The limit of sensitivity of the method is lower than 0.5 μg/ml of plasma or urine for each compound. The applicability of the method to pharmacokinetic studies of nalidixic acid in humans is demonstrated.