Effect of eating liquorice on the renin-angiotensin aldosterone axis in normal subjects.

The effect of confectionery liquorice on electrolyte status and the renin-angiotensin-aldosterone (RAA) axis was studied in 14 healthy volunteers. They ate liquorice in daily doses of 100 g or 200 g (equivalent to 0-7-1-4 g glycyrrhizinic acid) for one to four weeks. Plasma potassium concentrations fell by over 0-3 mmol/l in 11 people, including four who had to be withdrawn from the study because of hypokalaemia. One or more values of the RAA axis, especially plasma renin activity and urinary aldosterone concentrations, were considerably depressed in all subjects. These results show that potentially serious metabolic effects may occur in some people who eat modest amounts of liquorice daily for less than a week.     

Double column-switching high-performance liquid chromatographic method for the determination of TAK-603 and its metabolites in human serum

A double column-switching high-performance liquid chromatographic (HPLC) method for the determination of concentrations for TAK-603 (T) and its metabolites, T-72258 (M-I) and T-72294 (M-III), in human serum was developed. The analytes were extracted with ethyl acetate from human serum samples treated with triethylamine and injected into the HPLC system. Separation of the analytes was performed on the HPLC system with double column-switching technique. The mobile phases A and B for the first column and the mobile phase C for the second column used were a mixture of methanol–10 mM aqueous ammonium acetate solution (1:1, v/v), methanol and a mixture of methanol–10 mM aqueous ammonium acetate solution (11:9, v/v), respectively. The eluate was monitored with a UV detector at a wavelength of 253 nm. The work-up procedure was reproducible and more than 90% of the analytes could be recovered from human serum. The lower limits of quantitation were all 1 ng/ml for the analytes when 0.5 ml of human serum was used. Standard curves were linear with a correlation coefficient (R) of more than 0.999 in the range of 1–500 ng/ml for T, M-I and M-III in human serum. The intra- and inter-day precision of the method for the various analytes were below 4.8%. The accuracy was good with the deviations between spiked and calculated concentrations of the analytes being within 11.0%. The method was successfully applied to analyze serum samples after an oral administration of T to healthy male volunteers.     

Simultaneous determination of ascorbic acid, aminothiols, and methionine in biological matrices using ion-pairing RP-HPLC coupled with electrochemical detector

A novel highly sensitive ion-pairing reversed-phase high performance liquid-chromatography/electrochemical detection method for simultaneous determination of l-ascorbic acid, aminothiols, and methionine in biological matrices was developed, optimized, and validated. Reduced forms of the analytes were extracted from the sample matrices with 10% meta-phosphoric acid solution((aqueous)). To determine the total vitamin C, the total aminothiols, and the total methionine, samples were treated with tris(2-carboxyethyl)phosphine solution in 0.05% trifluoroacetic acid solution((aqueous)) subsequent to deproteination to reduce the oxidized forms of these compounds. Various analytes were separated on a C(18) (250 × 4.6 mm, 5 μm) analytical column using methanol-0.05% trifluoroacetic acid solution((aqueous)) (05/95, v/v), containing 0.1mM 1-octane sulphonic acid as the ion-pairing agent) as the isocratic mobile phase pumped at a flow rate of 1.5 mL min(-1) at room temperature. The column eluents were monitored at a voltage of 0.85 V. These analytes were efficiently resolved in less than 20 min using n-acetyl cysteine as the internal standard. The present method was specific for the analysis of these analytes and demonstrated acceptable values for linearity (r(2)>0.999 in the range of 0.2-10,000 ng mL(-1) for all the analytes), recovery (>96%), precision (%RSD ≤ 2.0), and sensitivity (on column limit of detection: 250-400 fg and limit of quantification: 0.8-1.25 pg), indicating that the proposed method could be efficiently used for determination of these analytes in the context of clinical research.     

Simultaneous quantification of tiloronoxim and tilorone in human urine by liquid chromatography-tandem mass spectrometry

A simple, sensitive and specific HPLC method with tandem mass spectrometry (HPLC/MS/MS) detection has been developed and validated for the simultaneous quantification of tiloronoxim and its major active metabolite, tilorone, in human urine. The analytes, together with metoprolol, which was employed as an internal standard (IS), were extracted with a mixture solvent of chloroform/ethyl ether (1/2, v/v). The chromatographic separation was performed on a narrow-bore reversed phase HPLC column with a gradient mobile phase of methanol/water containing 15 mM ammonium bicarbonate (pH 10.5). The API 3,000 mass spectrometer was equipped with a TurboIonSpray interface and was operated on positive-ion, multiple reaction-monitoring (MRM) mode. The mass transitions monitored were m/z 426.3-->100.0, m/z 411.3-->100.0 and m/z 268.3-->116.1 for tiloronoxim, tilorone and the IS, respectively. The assay exhibited a linear dynamic range of 1-100 ng/ml for both tiloronoxim and tilorone based on the analysis of 0.2 ml aliquots of urine. The lower limit of quantification was 1 ng/ml for both compounds. Acceptable precision and accuracies were obtained for concentrations over the standard curve ranges. Run time of 8 min for each injection made it possible to analyze a high throughput of urine samples. The assay has been successfully used to analyze human urine samples from healthy volunteers.     

Hollow fiber-based liquid phase microextraction (HF-LPME) for a highly sensitive HPLC determination of sulfonamides and their main metabolites

In this paper, three phase-hollow fiber-based liquid phase microextraction (HF-LPME) combined with a HPLC procedure using diode array (DAD) and fluorescence detection (FLD) has been developed for the determination of four widely used sulfonamides: sulfadiazine, sulfamerazine, sulfamethazine, sulfamethoxazole and their main metabolites, the corresponding N(4)-acetyl derivatives: N(4)-acetyl-sulfadiazine, N(4)-acetyl-sulfamerazine, N(4)-acetyl-sulfamethazine, N(4)-acetyl-sulfamethoxazole. A Q3/2 Accurel KM polypropylene hollow fiber supporting 1-octanol was used between a 2 M Na(2)SO(4) aqueous solution (pH 4) as a donor phase and aqueous solution (pH 12) as an acceptor phase. The procedure allows very low detection and quantitation limits of 0.3-33 ng L(-1) and 0.9-100 ng L(-1), respectively. The proposed method was applied to the determination of the analytes in environmental water samples (surface, tap and wastewater).     

Three phase liquid phase microextraction of phenylacetic acid and phenylpropionic acid from biological fluids

Three phase liquid phase microextraction (three phase LPME) technique coupled with HPLC-UV has been applied as a sensitive and efficient sample preparation method to determine phenylacetic acid (PAA) as a biomarker of depressive disorders and phenylpropionic acid (PPA) in biological fluids. The compounds were extracted from 3.0 ml aqueous solution with the adjustment of pH at a fixed value in the range of 2.0-3.5 (donor solution) into an organic phase (1-hexanol) layered on the surface of the donor solution and finally back-extracted into 4.0 microl of the acceptor microdrop (pH 11.1) located at the end of the microsyringe needle. After a prescribed back-extraction time, the acceptor microdrop was withdrawn into the microsyringe and then directly injected into the HPLC system. In order to achieve maximum extraction efficiency, different parameters affecting the extraction conditions were optimized. At the optimum conditions (donor solution: 2.3M Na(2)SO(4), pH 2.0-3.5; organic membrane: 95 microl of 1-hexanol; acceptor solution: 4.0 microl of 0.1M NH(3)/NH(4)(+) with pH 11.1; donor solution temperature: 45-50 degrees C; extraction time: 20 min and back-extraction time: 12 min), up to 110-fold enrichment factor was obtained. The calibration curve for these analytes was linear in the range of 1-5000 microg/l with r(2)>0.998. The intraday and interday RSD% were below 6.5% and the limits of detection (LODs) for both analytes were 0.2 microg/l (based on S/N=3). The proposed technique is a low cost, simple and sensitive method with highly clean-up effect. Finally, this technique was successfully utilized for the detection of target analytes in human urine, serum and plasma.     

Highly sensitive time-resolved fluorometric determination of estrogens by high-performance liquid chromatography using a beta-diketonate europium chelate

A new fluorescent europium chelate labeling reagent, 5-(4"-chlorosulfo-1',1"-diphenyl-4'-yl)-1,1,1,2,2-pentafluoro-3,5-pentanedione (CDPP), was synthesized for the time-resolved fluorometric detection of HPLC. The label can be directly bound to amino or phenolic hydroxyl groups of analytes with its chlorosulfonyl group, and the labeled analytes are separated on a HPLC column. After separation, EuCl(3), TOPO (tri-n-octylphosphine oxide), and Triton X-100 were added by post-column introduction to the eluent, and the fluorescence of the europium chelate was measured with the time-resolved fluorometric detector. Estrone (E1), 17beta-estradiol (E2), ethynylestradiol (EE2) and estriol (E3) were measured with the detection limits of 0.65, 0.65, 0.65 and 0.60 ng/ml, respectively. The recovery for river water samples was in the range of 86.0-105.1% with the RSD of 1.9-5.8%. The method was applied to the analysis of a river water sample and estrone (E1) was determined to be 2.1 ng/l. The results and processing have been compared with those of a GC-MS method and a high degree of correlation (r> or =0.98) was observed.     

Stable isotope-coded quaternization for comparative quantification of estrogen metabolites by high-performance liquid chromatography-electrospray ionization mass spectrometry

A fast and sensitive LC-ESI-MS method is described for the comparative quantification of 16 estrogen metabolites based on the derivatization of estrogens with a novel derivatizing reagent, N-methyl-nicotinic acid N-hydroxysuccinimide ester (C1-NA-NHS). The process introduces a quaternary amine to the analytes, making the analytes permanently charged regardless of the pH of the high-performance liquid chromatography (HPLC) mobile phase. This quaternization resulted in a highly efficient separation of 16 estrogen metabolites in 7 min at a detection level below 1 ng/mL. By using a deuterated derivatizing reagent (C1-d(3)-NA-NHS), a complete set of deuterated standards was utilized and used as internal standards in a comparative quantification and recovery study, demonstrating acceptable results over a wide concentration range. A pooled breast cancer serum sample was analyzed using the described method, and 15 estrogens were detected in the range of 80-530 pg/mL.     

Liquid chromatography-tandem electrospray mass spectrometry method for determination of serial chiral novel anticholinergic compounds of phencynonate in rat plasma

A sensitive and selective liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed for the determination of serial chiral novel anticholinergic compounds of phencynonate in rat plasma. After a simple protein-precipitation using methanol, the post-treatment samples were separated on a CAPCELL UG120 column with a mobile phase of a mixture of methanol and water (35:65) containing 0.1% formic acid. The serial chiral analytes and internal standard (IS) were all detected by the use of selected reaction monitoring mode (SRM). The method of all serial chiral analytes developed was validated in rat plasma with a daily working range of 0.5-100 ng/ml with correlation coefficient, R(2) > or = 0.99 and a sensitivity of 0.5 ng/ml as lower limit of quantification, respectively. This method was fully validated for the accuracy, precision and stability studies for all serial chiral analytes. The method proved to be accurate and specific, and was applied to the pharmacokinetic study of serial chiral novel anticholinergic compounds of phencynonate in rat plasma.     

Simultaneous liquid chromatographic determination of lamotrigine, oxcarbazepine monohydroxy derivative and felbamate in plasma of patients with epilepsy

A very simple and fast method has been developed and validated for simultaneous determination of the new generation antiepileptic drugs (AEDs) lamotrigine (LTG), oxcarbazepine's (OXC) main active metabolite monohydroxycarbamazepine and felbamate in plasma of patients with epilepsy using high-performance liquid chromatography (HPLC) with spectrophotometric detection. Plasma sample (500 microL) pre-treatment was based on simple deproteinization by acetonitrile. Liquid chromatographic analysis was carried out on a Synergi 4 microm Hydro-RP, 150 mm x 4 mm I.D. column, using a mixture of potassium dihydrogen phosphate buffer (50mM, pH 4.5) and acetonitrile/methanol (3/1) (65:35, v/v) as the mobile phase, at a flow rate of 1.0 mL/min. The UV detector was set at 210 nm. Calibration curves were linear (mean correlation coefficient >0.999 for all the three analytes) over a range of 1-20 mg/mL for lamotrigine, 2-40 microg/mL for monohydroxycarbamazepine and 10-120 microg/mL for felbamate. Both intra and interassay precision and accuracy were lower than 7.5% for all three analytes. Absolute recoveries ranged between 100 and 104%. The present procedure describes for the first time the simultaneous determination of these three new antiepileptic drugs. The simple sample pre-treatment, combined with the fast chromatographic run permit rapid processing of a large series of patient samples.     

Simultaneous determination of cefdinir and cefixime in human plasma by RP-HPLC/UV detection method: Method development, optimization, validation, and its application to a pharmacokinetic study

A novel isocratic reversed-phase high performance liquid-chromatography/ultraviolet detection method for simultaneous determination of cefdinir and cefixime in human plasma was developed and validated after optimization of various chromatographic conditions and other experimental parameters. Sample preparation based on a simple extraction procedure consisting of deproteination and extraction with 3 parts of 6% trichloroacetic acid aqueous solution followed by volume make up with the aqueous component of the mobile phase obtained best recoveries of the two analytes. Samples were separated on a Supelco Discovery HS C(18) (150 mm × 4.6 mm, 5 μm) analytical column protected by a Perkin Elmer C(18) (30 mm × 4.6 mm, 10 μm) guard cartridge. The mobile phase, methanol/acetonitrile (50/50, v/v):0.05% trifluoroacetic acid (19:81, v/v), operated at 50°C column oven temperature was pumped at a flow rate of 2.0 mL min(-1) and the column eluents were monitored at a wavelength of 285 nm. When Sample was injected into the Perkin Elmer high performance liquid-chromatography system through Rheodyne manual (or auto-sampler) injector equipped with 20 μL loop, separation was achieved within 4 min. The present method demonstrated acceptable values for selectivity, linearity within the expected concentration range (0.004-5.0 μg mL(-1); r(2)>0.999 for both analytes), recovery (>95% for cefdinir and >96% for cefixime), precision (%RSD<2.0 for cefdinir and <2.2 for cefixime), sensitivity (limit of detection: 1 ng mL(-1) and lower limit of quantification: 4 ng mL(-1) for both analytes), stability of solutions, and robustness. The method was efficiently applied to a pharmacokinetic study in healthy volunteers.     

Simultaneous determination of ten active components in traditional Chinese medicinal products containing both Gegen (Pueraria lobata) and Danshen (Salvia miltiorrhiza) by high-performance liquid chromatography

In order to facilitate the quality control of Traditional Chinese Medicine (TCM) products containing both Gegen (Pueraria lobata) and Danshen (Salvia miltiorrhiza), a new and simple HPLC method for the simultaneous determination of 10 active components in these products has been developed. The chromatographic separation was carried out on a C(18) column eluted with a mobile phase consisting of 0.1% acetic acid in water and 0.1% acetic acid in acetonitrile with gradient elution. The eluent was monitored by a photodiode array UV detector at a wavelength of 250 nm for Gegen components including puerarin, daidzein 8-C-apiosyl-glucoside, daidzin and daidzein, and at 270 nm for Danshen components including danshensu, protocatechuic aldehyde, salvianolic acid B, cryptotanshinone, tanshinone I and tanshinone IIa. Excellent chromatographic separation was achieved for all studied compounds with good linearity (r(2)> 0.999) over the studied concentration ranges. The developed method has been applied to the simultaneous determination of the 10 studied compounds in commercially available products containing both Gegen and Danshen. The TCM product samples were extracted by sonication with a mixture of methanol:water (80:20) containing 0.5% acetic acid. Extraction recoveries for all studied compounds were in the range of 96.01-106.18%. The intra-day and inter-day variations were less than 7.25 and 5.44%, respectively, for all studied compounds. The developed method has not only proved to be effective in the simultaneous determination of the 10 components, but also provides a convenient quality control approach for TCM products containing both Gegen and Danshen.     

Determination of D-amino acids labeled with fluorescent chiral reagents, R(-)- and S(+)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N, N-dimethylaminosulfonyl)-2,1,3-benzoxadiazoles, in biological and food samples by liquid chromatography

D-Amino acids in food and biological samples labeled with R(-)- and S(+)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N, N-dimethylaminosulfonyl)-2,1,3-benzoxadiazoles (DBD-PyNCS) were separated by reversed-phase chromatography and detected fluorometrically at 550 nm (excitation at 460 nm). DL-Amino acids were efficiently labeled at 55 degrees C for 20 min in basic medium. The resulting thiocarbamoyl-amino acids were resolved by an isocratic elution using water:30% methanol in acetonitrile (72:28) containing 0.1% trifluoracetic acid as mobile phase for hydrophilic amino acids and gradient elutions using sodium acetate buffer (pH 5. 2)/acetonitrile as gradient solvent mixture for hydrophobic amino acids, respectively. The detection limits (S/N = 3) of DL-amino acids tested were in the range of 0.16-0.75 pmol. The proposed method was applied to determine the D-amino acid(s) in milk, cream, fermented dairy products (yogurt and yakult), tomato products (juice, puree, and catchup), fermented beverages (beer and red wine), and human urine. The existence of D-amino acid(s) was demonstrated in all the samples tested. Furthermore, the identification of the D-amino acid(s) was performed using both isomers of DBD-PyNCS and by on-line HPLC-electrospray ionization-MS.     

Quantitative determination of the major saponin mixture bacoside A in Bacopa monnieri by HPLC

Bacoside A, the putative bioactive component of the Indian medicinal plant Bacopa monnieri, was found to be a mixture of saponins with bacoside A3 (1), bacopaside II (2), jujubogenin isomer of bacopasaponin C (3) and bacopasaponin C (4) as major constituents. An HPLC method together with an optimised extraction procedure was developed for the estimation of 1-4 in B. monnieri to enable standardisation of the latter. Concentration ranges of the analytes in samples of B. monnieri collected from different regions of India were 0.14-0.85% (w/w) (1), 0.12-0.69% (2), 0.05-0.72% (3) and 0.05-0.44% (4). The importance of using bacoside A, with known concentrations of 1-4, as a reference standard for the routine analysis of B. monnieri is highlighted. Two common flavonoids, luteolin and apigenin, were present in all samples of B. monnieri.     

Sensitive simultaneous determination of ciclesonide, ciclesonide-M1-metabolite and fluticasone propionate in human serum by HPLC-MS/MS with APPI

A new and very sensitive analytical method has been developed and validated to jointly determine the anti-inflammatory drug ciclesonide (CIC), its active principle metabolite M1 (CIC-M1) and fluticasone propionate (FP) in human serum, in the low concentration range from 10 to 1000 pg/mL. This was accomplished by high-performance liquid chromatography and tandem mass spectrometry using atmospheric pressure photo ionisation (HPLC-MS/MS with APPI) using 0.5 mL of serum. Serum was mixed with the internal standards (IS) D11-CIC and D11-CIC-M1 and extracted with diisopropylether. A gradient with acetonitrile (containing 10 mM of acetic acid and 10% of acetone) was used. HPLC-MS/MS of the acetic acid adducts of the analytes was performed in negative mode. The novel aspect of this method is that instead of the dopant being introduced directly into the source by means of an external HPLC pump, it was added to the mobile phase. This provided significantly better sensitivity than the usual method of in-source addition of the dopant, and with no loss in HPLC performance. Sensitivity for the analytes was about four times greater than with either APCI or ESI. Validation was performed in three batches. The inter-batch precision (CV) of the quality control samples in human serum ranged from 4.08% to 6.78% for CIC, from 2.57% to 7.74% for CIC-M1, and from 2.38% to 9.61% for FP. The inter-batch accuracy (with reference to the mean value) of the quality control samples in human serum ranged from 99.3% to 110.0% for CIC, from 101.8% to 104.7% for CIC-M1, and from 100.4% to 101.8% for FP. Calibration data and LLOQ data are also presented in this paper. The analytes were stable in human serum over three freeze/thaw cycles, or for 4h at room temperature, or for at least 18 months when stored at below -20 degrees C. This method was used for quantifying the analytes after inhalation of low-mug amounts of the drugs by patients.     

Validation of an HPLC-UV method according to the European Union Decision 2002/657/EC for the simultaneous determination of 10 quinolones in chicken muscle and egg yolk

Herein two different methods are proposed for the determination of 10 quinolones (enoxacin, ofloxacin, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin, oxolinic acid, nalidixic acid and flumequine) in chicken muscle and egg yolk. Two different HPLC systems were used comparatively and the respective methods were fully validated. The analytes were initially extracted from chicken muscle and egg yolk and purified by a solid phase extraction using LiChrolut RP-18 cartridges. Recoveries varied between 96.6 and 102.8% for chicken muscle and 96.4-102.8% for egg yolk. HPLC separation was performed at 25 degrees C using an ODS-3 PerfectSilTarget (250 mmx4 mm) 5 microm analytical column (MZ-Analysentechnik, Germany). The mobile phase consisted of a mixture of 0.1% trifluoroacetic acid (TFA)-ACN-CH3OH, delivered by a gradient program, different for each method. In both cases caffeine was used as internal standard at the concentration of 7.5 ng/microL. Column effluent was monitored using a photodiode array detector, set at 275 and 255 nm. The developed methods were validated according to the criteria of Commission Decision 2002/657/EC. The LODs for chicken muscle varied between 5.0 and 12.0 microg/kg and for egg yolk was 8.0 microg/kg for all examined analytes.     

Simultaneous determination of urinary cortisol, cortisone and corticosterone in parachutists, depressed patients and healthy controls in view of biomedical and pharmacokinetic studies

A rapid and sensitive reversed-phase liquid chromatographic method (RP-LC) with UV detection has been developed for the determination of free cortisol, cortisone and corticosterone in human urine. The assay was performed after a solid-phase extraction procedure (SPE) with dexamethasone as the internal standard. Chromatographic separation was carried out on a Nucleosil 100 C(18) analytical column using a mixture of acetonitrile and water (30 : 70, v/v) as a mobile phase at a flow-rate of 1 mL min(-1). Spectrophotometric detection was performed at 240 nm. The method has been validated for accuracy, precision, selectivity, linearity, recovery and stability. The absolute recoveries of glucocorticoids were above 94.6%. The limits of detection (LOD) and quantification (LOQ) were 0.5 and 2 ng mL(-1), respectively, for all analytes. Linearity was confirmed in the range of 2-300 ng mL(-1) with a correlation coefficient greater than 0.9997 for all steroid hormones. The proposed method was sensitive, robust and specific allowing reliable quantification of steroid hormones. This method was successfully applied for determination of three endogenous glucocorticoid levels in human urine. The studies were performed on 20 sedentary healthy volunteers in comparison to two socially diversified groups, namely 10 parachutists before and after jump and 10 patients with depression. Pharmacokinetic studies performed on these groups indicated that urinary free cortisol and cortisol-to-cortisone ratios can be treated as biomarkers of stress and depressive disorders.     

MalphaNP acid, a powerful chiral molecular tool for preparation of enantiopure alcohols by resolution and determination of their absolute configurations by the (1)H NMR anisotropy method

A novel methodology using a chiral molecular tool of MalphaNP acid (1), 2-methoxy-2-(1-naphthyl)propionic acid, useful for preparation of enantiopure secondary alcohols and determination of their absolute configurations by the (1)H NMR anisotropy method was developed; racemic MalphaNP acid (1) was enantioresolved with (-)-menthol, and the enantiopure MalphaNP acid (S)-(+)-(1) obtained was allowed to react with racemic alcohol, yielding a mixture of diastereomeric esters, which was clearly separated by HPLC on silica gel. By applying the sector rule of (1)H NMR anisotropy effect, the absolute configuration of the first-eluted MalphaNP ester was unambiguously determined. Solvolysis or reduction of the first-eluted MalphaNP esters yielded enantiopure alcohols.     

Determination of modafinil, modafinil acid and modafinil sulfone in human plasma utilizing liquid-liquid extraction and high-performance liquid chromatography

An assay was developed to determine concentrations of modafinil (dl-2-[(diphenylmethyl)sulfinyl]acetamide; Provigil) and its two major circulating metabolites, modafinil acid and modafinil sulfone, in human plasma. The assay utilized liquid-liquid extraction of the analytes and an internal standard, (phenylthio)acetic acid, from plasma into a mixture of hexane-dichloromethane-glacial acetic acid (55:45:2, v/v). The analytes were resolved isocratically on a narrow-bore phenyl column at a mobile phase flow-rate of 0.3 ml/min and were monitored by UV detection at 235 nm. The method reported herein reduces the required sample volume of previously reported methods from 1.00 to 0.200 ml of plasma while lowering the limit of quantification (LOQ). The linear range of the assay was from 0.100 to 20.0 microg/ml for each of the three compounds.