Mass versus molar doses, similarities and differences.

Generally, they are two systems expressing the amounts of active substance in a given drug product, i.e. mass and molar dose. Currently, the dose system based on the mass is widely used in which doses are expressed in grams or milligrams. On the other hand, the molar dose system is in direct relation to the number of molecules. Hence, the objective of this work was to compare both systems in order to find their advantages and disadvantages. Active substances belonging to the groups of antibiotics, nootropic agents, beta-blockers, vitamins, GABA-analog, COX-2 inhibitors, calcium channel antagonists, benzodiazepine receptor agonists, lipid-modifying agents (fibrates), non-steroidal anti-inflammatory drugs (profens), estrogens, neuroleptics, analgesics and benzodiazepines were considered. Moreover, products containing two active substances were also taken into account. These are mixtures of hydrochlorothiazide with active substances influencing the renin-angiotensin system and combined oral contraceptives. For each active substance, belonging to the groups mentioned above molar doses were calculated from mass doses and molar mass. Hence, groups of drugs with a single active substance, drugs with similar pharmacological activities, pharmaceutical alternatives, and drugs with a single active ingredient manufactured in different doses were compared in order to find which dose system describes more adequately differences between and within the groups mentioned above. Comparisons were supported by a number of equations, which theoretically justify the data, and relationships derived from calculations.

Validated method for L-ornithine-L-aspartate analysis in human plasma by capillary electrophoresis.

A rapid capillary electrophoresis method for routine determination of two amino acids, L-ornithine and L-aspartic acid, in human plasma is reported. The method runs automatically, requires a minimum of sample preparation and moreover includes no extensive extraction and no gradient or derivatization procedure. Analyses were performed on an uncoated silica capillary using buffer solution composed with 10 mM sodium tetraborate and 1 M sodium hydroxide (pH=10.0). A capillary electrophoresis P/ACE system equipped with UV detection (200 nm), an automatic injector, a fluid cooled cartridge and System Gold data station was used in this study. The total analysis time under these conditions was 8.0 min. The calibration curve was linear in the range 10-280 microg mL-1 for L-aspartic acid and 20-280 microg mL-1 for L-ornithine (for both amino acids, r=0.999). The method was validated by inaccuracy (bias) and precision (RSD) studies by analysing samples. The method was successfully applied to the quantitative determination of L-ornithine-L-aspartate in human plasma and could be useful for clinical and bioavailability investigations.

Capillary electrophoresis in analysis of veterinary drugs.

Antibiotics are extensively applied in veterinary medicine for the treatment of various bacterial infections. Because of their use in food producing animals, the risk of occurrence of unwanted residues in edible products exists. To ensure human food safety, The European Union has defined maximum residue limits (MRLs) for veterinary drug residues in food products. Analytical methods need to be developed to confirm the presence of antibiotics at the MRL level. A capillary electrophoresis (CE) method with UV detection is proposed for the quantitative determination of residues from poultry and porcine tissues. Eight of the most frequently used antibiotics and nifursol, which routinely used as poultry coccidiostat, were analysed. CE technique permitted analysing substances to be separated from muscle, liver, kidney and skin with fat after a simple extraction with acetonitrile or ethyl acetate under basic conditions. Proposed method is capable to identify drug residues in tissues at level below 20 microg/kg.

RP-HPLC method with electrochemical detection for the determination of metoclopramide in serum and its use in pharmacokinetic studies.

A rapid and sensitive reversed-phase high performance liquid chromatographic method has been developed for the determination of metoclopramide in serum. The assay was performed after single extraction with ethyl ether using methyl parahydroxybenzoate as internal standard. Chromatographic separations were performed on C(18) stationary phase with a mobile phase composed of methanol-phosphate buffer pH 3 (30:70 v/v). Analytes were detected electrochemically. The quantification limit for metoclopramide in serum was 2 ng mL(-1). Linearity of the method was confirmed in the range of 5-120 ng mL(-1) (correlation coefficient 0.9998). Within-day relative standard deviations (RSDs) ranged from 0.3 to 5.5% and between-day RSDs from 0.8 to 6.0%. The analytical method was successfully applied for the determination of pharmacokinetic parameters after ingestion of 10 mg dose of metoclopramide. Studies were performed on 18 healthy volunteers of both sexes.

Retention and separation studies of cholesterol and bile acids using thermostated thin-layer chromatography.

The influence of temperature on retention and separation of cholesterol and bile acids, using reversed-phase thin-layer chromatography, was studied. As mobile phases methanol-water mixtures of various compositions were used. Chromatographic experiments were performed using vapor-saturated chambers at temperatures ranging from 5 to 60 degrees C. A linear relationship between R(M) values and temperature (1/T) as well as mobile phase composition was observed. The elution order of steroids under the conditions investigated was discussed. Each chromatogram was evaluated using simple optimization parameters and the best chromatographic conditions for the separation of multicomponent samples were chosen.

The equilibrium constant of beta-cyclodextrin-phenolphtalein complex; influence of temperature and tetrahydrofuran addition.

The temperature influence on creation of a supramolecular complex in which beta-cyclodextrin (beta-CD) is the host molecule and phenolphtalein (PP) is the guest has been studied in aqueous solution by UV-visible absorption spectroscopy. The decrease of temperature of beta-cyclodextrin-phenolphtalein system resulted in a decrease in absorbance of the UV-vis spectrum. Under favourable conditions (0.1 mM beta-CD, 30 microM PP) the termochromic effect is very significant (approximately =0.1 U of absorbance/10 degrees C). The formation constant of inclusion complex was determined at various temperatures (from 10 to 70 degrees C) using Scott's equation. The association constants (K11) for the binding in 0.02 M sodium carbonate (pH 10.5) at 10 and 70 degrees C are 7.44 and 0.26 x 10(4) M(-1) respectively. The stoichiometric ratio of investigated complex was found to be 1:1 on wide range of beta-cyclodextrin:phenolphtalein concentration ratio (from 0.8:1 to 427:1). Additionally, strong interaction between cyclodextrin and tetrahydrofuran (THF) was observed and the inhibitory effect of tetrahydrofurane on the association of beta-CD PP complex was studied. From linear Van't Hoff plots thermodynamic parameters such as: the change of enthalpy (deltaH(o)) and change of entropy (deltaS(o)) were estimated and interpreted.

Determination and pharmacokinetics of acyclovir after ingestion of suspension form.

The study describes, simple, precise, sensitive and accurate HPLC assay with spectrofluorimetric detection for the determination of acyclovir in human plasma. The method was linear over a range 25 1200 ng ml(-1). The average yield in this method exceeded 80%. Limits of quantitation and detection were 25 and 10 ng ml(-1), respectively. On the basis of reported method, a single-dose of pharmacokinetics on 24 men, in two doses (200 and 400 mg) of acyclovir suspension has been investigated. Pharmacokinetic parameters obtained from both doses of the drug were compared. The linearity of acyclovir pharmacokinetics in the investigated dose ranges has been confirmed.

The influence of temperature on the multiple separation of estrogenic steroids using mobile phases modified with beta-cyclodextrin in high-performance liquid chromatography.

The effect of temperature on the retention and multiple separation of six estrogenic steroids in reversed-phase liquid chromatography has been studied. Capacity factors (k') of estriol, 17 beta-estradiol, 17 alpha-estradiol, d-equilenin, equilin and estrone were measured using mobile phase modified with different concentrations of beta-cyclodextrin (from 0-16 mM), a fixed solvent composition (acetonitrile-water) and a wide range of column temperatures (from 5 to 80 degrees C). The plots of capacity factors vs. reciprocal of absolute temperature are nonlinear in each case when mobile phase modified with beta-cyclodextrin was used. Particularly strong nonlinearity was observed at lower temperature and at higher beta-cyclodextrin concentration. The complex chromatograms were evaluated using optimization parameters such as capacity factor of the last-eluted peak (k'max), the smallest resolution between adjacent peaks (Rs,min) and relative resolution product (r). The results presented describe precisely the role of temperature in high-performance liquid chromatography systems in which mobile phases modified with cyclodextrin were used. Moreover, the elution order of estrogenic steroids on modified and unmodified mobile phases has been discussed.

Determination and pharmacokinetics of a furosemide-amiloride drug combination.

The study presents an accurate and precise HPLC assay for the determination of furosemide and amiloride in human specimens. Both drugs were extracted from human plasma with ethyl acetate; furosemide was extracted at pH 1 and amiloride at pH 12. While chromatographic separation conditions, i.e., column, mobile phase and flow-rate were the same for both investigated drugs, furosemide was detected using a UV absorbance detector, whereas amiloride, because of its very low therapeutic range, was detected with a spectrofluorimetric detector. The linearity of the furosemide and amiloride assays were confirmed over the range of 30-3000 ng/ml and 0.5-30 ng/ml, respectively. These concentrations correspond well with the therapeutic ranges of both drugs. The extraction recoveries, depending on concentration, exceed 80% for furosemide and 74% for amiloride. The reported methods were applied to pharmacokinetic investigations of the two compounds taken in form of a drug combination.

Determination of piracetam in human plasma by capillary electrophoresis.

A capillary electrophoresis (CE) procedure has been developed for the determination of piracetam in human plasma. Analyses were performed on an uncoated silica capillary using borax buffer modified with the addition of alpha-cyclodextrin. The detection was UV, operated at 200 nm. The detection limit of the authentic samples was 1 microg/ml. The calibration curve was linear over a range of 4 to 24 microg/ml (r=0.997). Inter-assay R.S.D. was below 9.3%. The described method has been successfully applied to the quantitative determination of piracetam in human plasma and should be useful for clinical and bioavailability investigations.

The influence of temperature on the high performance liquid chromatographic separation of steroids using mobile phases modified with beta-cyclodextrin.

The effect of temperature on the retention and multiple separation of hydrocortisone, testosterone, 17 alpha-methyltestosterone, prednisone, cortisone and 17 alpha-hydroxyprogesterone in reversed-phase liquid chromatography has been studied. Capacity factors (k') of the steroids were measured using a mobile phase modified with different concentrations of beta-cyclodextrin (from 0-16 mM), a fixed solvent composition (acetonitrile-water) and a wide range of column temperatures (from 5-80 degrees C). The plots of capacity factors vs. reciprocal of absolute temperature are nonlinear in every case when mobile phase modified with beta-cyclodextrin was used. Particularly strong nonlinearity was observed at lower temperature and at higher beta-cyclodextrin concentration. The complex chromatograms were evaluated using optimization parameters such as capacity factor of the last-eluted peak (k'max), the smallest resolution between adjacent peaks (Rs; min) and relative resolution product (r). The results presented describe precisely the role of temperature in high performance liquid chromatography systems in which mobile phases modified with cyclodextrin were used.

Simultaneous determination of spironolactone and its metabolites in human plasma.

This study describes a specific, precise, sensitive and accurate method for determination of unchanged spironolactone and its major active metabolites in human plasma. After one-step liquid-liquid extraction, analysis of the parent drug and its metabolites was performed in one chromatographic run, using a high performance liquid chromatography (HPLC) method with a programmed switchover of the UV wavelength. Spironolactone and 7 alpha-thiomethyl-spironolactone were detected at 245 nm, while canrenone and internal standard were detected at 280 nm. The column used was an S5 ODS2 (500 mm x 4.6 mm i.d.). The mobile phase was a mixture of acetonitrile-aqueous orthophosphoric acid (pH 3.4). Chromatographic separations were performed at 5 degrees C. The standard curves were linear over the range 10-400 ng ml-1 for spironolactone and 10-600 ng ml-1 for 7 alpha-thiomethyl-spironolactone and canrenone. The precision and accuracy of the method were confirmed by relative standard deviations below 10% for different concentrations, except for the concentration equal to the quantitation limit, where these parameters ranged from 12-15%. The recovery was above 80% for all investigated compounds and for the internal standard. The assay proved to be suitable for pharmacokinetic studies of spironolactone.

High-performance liquid chromatographic and capillary electrophoretic determination of free nicotinic acid in human plasma and separation of its metabolites by capillary electrophoresis.

Two methods are described based on high-performance liquid chromatography and capillary electrophoresis that provide the selective and sensitive determination of nicotinic acid in human plasma. Moreover, the capillary electrophoresis system was used for the separation of nicotinic acid, nicotinamide, nicotinamide N-oxide, N'-methylnicotinamide, 6-hydroxynicontinic acid, nicotinuric acid and barbital (internal standard). The extraction procedure is simple; no gradient elution or derivatization is required. Both methods can be useful for clinical and biomedical investigations.

Effect of temperature on separation of estradiol stereoisomers and equilin by liquid chromatography using mobile phases modified with beta-cyclodextrin.

The unusual temperature effect on the retention of 17 alpha-estradiol, 17 beta-estradiol and equilin in reversed-phase liquid chromatography has been observed. Capacity factors (k') of the steroids were measured using mobile phase with different concentrations of beta-cyclodextrin (from 0 to 16 mM) in a fixed mobile phase composition (acetonitrile-water) and wide range of column temperatures (from 5 degrees to 80 degrees C). The plots of capacity factors vs reciprocal of absolute temperature are nonlinear in each case. At subambient temperatures the capacity factors decreased with temperature decrease. This effect is more evident for the natural 17 beta-estradiol than for its 17 alpha-isomer.

Captopril: determination in blood and pharmacokinetics after single oral dose.

The study describes a specific, precise, sensitive and accurate method for determination of unchanged captopril, an angiotensin-converting enzyme inhibitor, in human plasma. Captopril was stabilized by forming an adduct with p-bromophenacyl bromide and this adduct was measured by high-performance liquid chromatography with UV detection. The standard curve was linear over a range of 30-800 ng ml-1. The average yield of derivatization of the unchanged captopril was 73.6% and the recovery of captopril-adduct reached 93.1%. The limit of detection was 15 ng ml-1, while the quantitative limit was 30 ng ml-1. Inter- and intra-assay RSD was below 9%, but inter- and intra-assay accuracy was below 8%. On the basis of elaborated method, a single-dose pharmacokinetics in 12 men, in two doses (25 and 50 mg of captopril) has been investigated. The comparison of the pharmacokinetic parameters obtained from both doses of the drug have been made.

Effect of temperature on separation of norgestrel enantiomers by high-performance liquid chromatography.

The influence of mobile phase composition, concentration of beta-cyclodextrin and temperature on the high-performance liquid chromatographic separation of norgestrel was studied. In studies of the effect of temperature on the enantioselectivity of (+/-)-norgestrel, acetonitrile-water (25:75, v/v) modified by the addition of beta-cyclodextrin (14 mM) was applied as the mobile phase. Enantiomers were detected using UV detection at 240 nm. The capacity factors were measured over a wide range of column temperatures from -5 to 70 degrees C.

Evaluation of chromatographic methods for the determination of nifedipine in human serum.

Gas-liquid chromatography and high-performance liquid chromatography were compared for the identification and determination of nifedipine in biological samples and the elaboration of the optimum liquid-liquid extraction procedure. The determination limits were 2 and 10 ng/ml, respectively, and the detection limits were 1 and 5 ng/ml, respectively. The calibration graphs were linear in the ranges 2-300 and 10-500 ng/ml, respectively. Recoveries based on three different concentrations were 88.7-95.8% and 93.7-104.2%, respectively. Both methods are sensitive, specific and reproducible enough for pharmacokinetic studies and therapeutic drug monitoring.

Chromatographic studies of the enantiomeric composition of some therapeutic compositions applied in the treatment of liver and kidney diseases.

A gas-liquid chromatographic system with alpha-cyclodextrin in formamide medium (coated on Chromosorb) was used for the separation of enantiomers of alpha-pinene, beta-pinene, limonene and camphene in medicines applied in the therapy of liver and kidney diseases. The drugs under investigation were produced in Poland (Terpichol and Terpinex), in Germany (Rowachol and Rowatinex) and in Slovenia (Uroterp). It was found that, depending on the manufacturer, medicines possessing similar chemical compositions differ considerably from one another regarding the content of enantiomers, mainly those of alpha-pinene.

Fluorescence enhancement of two terpenes commonly present in essential oils.

The fluorescence spectra of anethole and eugenol dissolved in methanol-aqueous binary systems with the addition of alpha- and beta-cyclodextrin were studied. Observed enhancement of the fluorescence intensity is possibly due to the higher quantum yield of the cyclodextrin-hydrocarbon inclusion complexes. The measured fluorescence intensities for eugenol and anethole in the presence of alpha- and beta-cyclodextrin were processed using principal component analysis. The results obtained suggest 1:1 and 2:1 complexation, as confirmed by a double reciprocal plot for terpenes complexed to cyclodextrins. In both cases (anethole and eugenol) detection limits were improved after addition of cyclodextrins. This phenomenon can be applied for improvement of direct fluorescence and HPLC-fluorescence assays.