Evaluation of alpha-methylacyl-CoA racemase, metallothionein and prostate specific antigen as prostate cancer prognostic markers.

Current diagnostic techniques are inefficient in distinguishing latent and low-risk forms of prostate cancer from high-risk forms. The present study is focused on determination of putative tumor markers of aggressive high-grade forms of prostate cancer. Potential markers were determined in blood sera of 133 patients (82 cases and 51 controls) and in cell lines (Gleason score 9-derived 22Rv1 and normal tissue derived PNT1A) on mRNA and protein levels. Alpha-methylacyl-CoA racemase (AMACR), metallothionein classes 1A and 2A (MT1A and MT2A) were determined and compared to prostate specific antigen (PSA) levels. On mRNA level, significantly increased expression of MT2A (2.4-fold), PSA (2.6-fold) and AMACR (8.4-fold) and insignificantly (1.9-fold) elevated MT1A in 22Rv1 compared to non-tumor PNT1A were determined. On protein level, significant enhancement of free PSA and total PSA in tumor cell line was evident. AMACR protein was 1.5-fold elevated in tumor line (below the level of significance). Contrary to mRNA, significantly (p = 0.01) reduced level of MT protein in tumor lines was determined. In the case of serum level, significantly enhanced MT level (4.5-fold) in patients' sera was found. No significant changes were observed in the case of AMACR. These findings indicate possible alternative role of MT to PSA prostate cancer marker. In addition, level of AMACR is distinctly higher in the Gleason score 9 in serum of patients and MT shows a descending trend in relation to Gleason score.

Relationship between haematological profile and progression or spontaneous regression of melanoma in the Melanoma-bearing Libechov Minipigs.

Haematological parameters, plasma iron concentration, and bodyweight were monitored in Melanoma-bearing Libechov Minipigs (MeLiM) from 5 to 18 weeks old. Animals with melanoma progression (P group) and spontaneous regression (SR group) were compared. The P group showed the lowest median values of red blood cell counts (RBC), haematocrit (HCT), haemoglobin concentration (HGB), and bodyweight, whereas the control white (tumour-free) pigs (C group) revealed the highest mean values of these parameters. The mean values of pigs with SR fell between the P and C groups. In addition, a stable concentration of plasma iron was found in the C group, while iron deficiency that increases with age was observed in the MeLiM groups. These results indicate that MeLiM are affected by cancer-related microcytic hypochromic anaemia. The lowest values of HGB, RBC, and HCT, together with the highest number of platelets (PLT) in the P group correspond to melanoma progression. Higher values of these parameters and lower PLT in the MeLiM pigs with SR reflected health improvement due to the destruction of melanoma cells during spontaneous regression. Monitoring of these haematological parameters can help distinguish MeLiM piglets with progression and spontaneous regression of melanoma in the early stages of postnatal development. The findings of this study correspond to findings in human patients in which cancer-related anaemia, thrombocytosis, and iron deficiency are often diagnosed.

Optimization of HPLC chromatographic conditions for determination of Transkarbam 12 and its degradation products.

This paper deals with searching of HPLC chromatographic conditions for determination and separation of Transkarbam 12 (T 12) and its two main degradation products (omega-aminocaproic acid and dodecylalcohol). T 12 is a new substance which belongs to the group of accelerators of transdermal penetration. Chromatographic separation was achieved using Separon SGX C18 analytical column (150 mm x 3 mm i.d.; 5 microm). Mobile phase contained acetonitrile and sodium acetate buffer pH 4.5 at the flow rate of 1 ml/min. Separation was carried out under the conditions of gradient elution. After the modification of the structure by derivatization reagent (3,5-dinitrobenzoyl chloride) detection at wavelength 230 nm was realized. The aim of this study was not only the optimization of the separation of derivatization reagent and derivatized T 12, Ak and D but also optimal derivatization processes for all three substances.

Use of the zirconia-based stationary phase for separation of ibuprofen and its impurities.

A new reversed-phase liquid chromatographic method using zirconia-based stationary phase was developed for determination of ibuprofen, its related compounds and its main degradation products. The chromatographic separation was successfully achieved on the Discovery Zr-PS column (150 mm x 4.6 mm i.d., 5 microm), using a mobile phase methanol-phosphate buffer (pH 4.5; 0.05 M)-tetrahydrofurane (21:74:5, v/v/v) and the flow rate 0.5 ml min(-1). The UV detection was performed in dual wavelength mode (219 and 258 nm) to detect all compounds of interest. The column temperature was set on 60 degrees C to shorten the analysis time and improve the peak symmetry. The method is simple, rapid and cuts down the amount of hazardous waste produced in the analysis. The assay is completed within 22 minutes.

HPLC evaluation of diclofenac in transdermal therapeutic preparations.

High-performance liquid chromatography (HPLC) was selected for analytical evaluation of sodium diclofenac in original transdermal therapeutic preparations containing adjuvant substances (capsaicin, hyoscyamine). After isolation from laminated adhesive patches, diclofenac was analysed on columns with reversed phase, using the mobile phase ethanol and phosphate buffer (pH 6.5) with an addition of tetrabutylammonium iodide and detection at 284 nm. Not only the total amount of diclofenac in the patch was evaluated, but HPLC methodology was also employed to select a suitable acceptor medium for permeation experiments. In patches manufactured in the tested series, HPLC was also employed to examine the release of diclofenac and its in vitro permeation through the human skin.

Determination of ibuprofen in erythrocytes and plasma by high performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method is described for the determination of ibuprofen in isolated erythrocytes and plasma. Before HPLC analysis ibuprofen was isolated by liquid-liquid extraction from these biological matrices; methylene chloride proved to be the best of the organic solvents tested. For the sample of erythrocytes it was necessary to carry out haemolysis prior to their extraction. HPLC was performed on a C-18 column with a mobile phase of methanol-water (220:100, v/v) acidified with perchloric acid to pH 3. Ultraviolet detection was at 222 nm. This method has been applied to the quantification of ibuprofen in rabbit erythrocytes and plasma for a pharmacokinetics study.

Solid-phase extraction and HPLC analysis of kebuzone and its metabolites in blood.

A HPLC method for quantification of kebuzone and its metabolites in whole blood was developed. The compounds and the internal standard were isolated from blood by solid-phase extraction on a C-18 cartridge. A blood sample was to be hemolyzed before extraction. HPLC was performed on a C-18 column with the mobile phase composed of methanol/water acidified to pH 2.7 and UV absorbance detection at 247 nm. This method has been successfully applied to a pharmacokinetic study of kebuzone and its metabolites in rabbits.

HPLC evaluation of drugs in transdermal therapeutic preparations. I. Glycerol trinitrate.

In connection with the development of transdermal delivery systems (TTS) of the laminated adhesive patches type, HPLC was selected for analytical evaluation of active principles. It was employed for analysis of glycerol trinitrate (GTN) as one of the drugs administered in the form of medicinal adhesive patches. After isolation from the patch, GTN was analysed by reverse phase HPLC, employing methanol and water as the mobile phase, and detected at 206 nm. The total amount of GTN in the patch was evaluated and its concentrations in a lactose trituration and the reservoir layer were determined. In the patches manufactured in a test series, the elaborated HPLC method was used to investigate the release of GTN in a liberation study and its permeation through excised human skin in experiments performed in vitro. In connection with the stability study, the decomposition products of GTN were also determined.

[HPLC analysis of non-steroidal antirheumatic agents in biological material. I. Salicylates]. / HPLC analýza nesteroidních antirevmatik v biologickém materiálu I. Salicyláty.

The present paper surveys both published HPLC methods estimating salicylates in biological materials and an HPLC method for estimation of salicylic acid and its metabolites in the samples of whole blood, isolated erythrocytes and plasma. For liquid-liquid extraction, salicylates were analyzed on reverse phases Separon SGX C18 with the mobile phase methanol-water (pH 2,5) and detected at 236 nm. The elaborated method was used in a pharmacokinetic study of salicylates on rabbits.

[HPLC analysis of non-steroidal antirheumatic agents in biological material. II. Profens]. / HPLC analýza nesteroidních antirevmatik v biologickém materiálu II. Profeny.

The present paper surveys the published HPLC methods evaluating profens in biological materials and reports a HPLC method for the determination of ibuprofen in the samples of whole blood. For liquid-liquid extraction, ibuprofen was analyzed on the reverse phases Separon SGX C18 with the mobile phase methanol-water (pH 3.0) and detected at 222 nm. The elaborated method was employed in a pharmacokinetic study of ibuprofen on laboratory rabbits.

[HPLC analysis of nonsteroidal antirheumatic agents in biological materials. III. Pyrazolidinediones]. / HPLC analýza nesteroidních antirevmatik v biologickém materiálu III. Pyrazolidindiony.

The present paper surveys published HPLC methods estimating pyrazolidinediones in biological materials and an HPLC method for estimation of kebuzone in the samples of whole blood, isolated erythrocytes and plasma. An addition of propyl gallate prevented undesired oxidation of kebuzone. For liquid-liquid extraction, kebuzone was analysed on the reverse phases Separon SGX C-18 with the mobile phase methanol--water (pH 2.7) and detected at 247 nm. The elaborated method was used in a pharmacokinetic study of kebuzone on rabbits.

[HPLC analysis of nonsteroidal antirheumatic substances in biological material. V. Fenamates]. / HPLC analýza nesteroidních antirevmatik v biologickém materiálu V. Fenamáty.

The present paper surveys published HPLC methods estimating derivates of fenamic acid in biological materials and an HPLC method for estimation of meclofenamic acid in the samples of whole blood. Meclofenamic acid was isolated from the sample of the blood both liquid-liquid extraction and solid-liquid extraction. For determination of meclofenamic acid in the blood was preferred solid-liquid extraction. The adjust sample was analysed on reverse phase Separon SGX C-18 with the mobile phase methanol-water (pH 2.3) and detected at 220 nm. The method was used in a pharmacokinetic study of meclofenamic acid on rabbits.

[HPLC analysis of nonsteroidal antirheumatic agents in biological materials. IV. Acetic acid derivatives]. / HPLC analýza nesteroidních antirevmatik v biologickém materiálu. IV. Fenaky.

The present paper surveys the published HPLC methods estimating derivatives of acetic acid in biological materials and an HPLC method for estimation of indomethacine in the samples of whole blood. The elaborated chromatographic conditions and process of isolation were applied also to the analysis of tropesine and both drugs were determined simultaneously. The blood samples were adjusted by liquid-liquid extraction and indomethacine (and tropesine) was analysed on the reverse phase Separon SGX C-18 with the mobile phase methanol-water (pH 3.2) and detected at 254 nm. The method was used in a pharmacokinetic study of indomethacine on rabbits.

An innovative approach to the analysis of 3-[4-(2-methylpropyl)phenyl]propanoic acid as an impurity of ibuprofen on a carbon-coated zirconia stationary phase.

3-[4-(2-Methylpropyl)phenyl]propanoic acid has been introduced as impurity F to the European Pharmacopoeia in its Supplement 4.2. In contrast to other impurities, which are evaluated by HPLC, the content of impurity F is determined by gas chromatography after previous derivatization. Thus a novel reversed-phase HPLC method was developed to simplify the evaluation of pharmacopoeial impurity F of ibuprofen. Favourable properties of zirconia stationary phases were employed for this purpose. The HPLC separation was achieved on a Zr-CARB column (150 mm x 4.6mm i.d., 5 microm) using the mobile phase acetonitrile-phosphate buffer (pH 3.5, 25 mM) (38:62, v/v), temperature 80 degrees C and the flow rate 1.2 ml min(-1). The fluorescence detection was employed to enhance the sensitivity of the method. Optimal detection parameters were chosen on the basis of fluorescence spectra of the analytes. The excitation and emission wavelengths were 220 nm and 285 nm, respectively. The analysis was completed within 25 min. The subsequent validation of the method confirmed the applicability of method for the analytical assay of impurity F.

High-performance liquid chromatographic determination of terguride in solid dosage forms and plasma.

A simple and rapid HPLC method was developed to determine terguride in terguride hydrogenmaleate, coated tablets and plasma. The assay was carried out on a glass column of SGX CN (150 x 3.3 mm I.D.) using methanol and phosphate buffer solution (pH 7.0) as the mobile phase, with detection at 227 nm. Terguride was quantified using promethazine as an internal standard. The tablet matrix was extracted into methanol. Plasma samples were deproteinated with acetonitrile and the supernatant was injected into the HPLC system. The method is linear, quantitative and reproducible.

High-performance liquid chromatographic assay for ibuprofen in whole blood using solid-phase extraction.

A precise, accurate, reproducible one-step method for the high-performance chromatographic determination of ibuprofen in whole blood is described. Samples were, after haemolysis, prepared by solid-phase extraction. Analyses were performed using reversed-phase chromatography on a Separon SGX C18 column with a mobile phase of methanol-water (pH 3) and ultraviolet detection at 222 nm. The method was used for pharmacokinetic studies in rabbits.

Simultaneous high-performance liquid chromatographic determination of salicylates in whole blood, plasma and isolated erythrocytes.

A method using liquid-liquid extraction has been developed for the isolation of acetylsalicylic acid and its metabolites, salicylic, gentisic or possibly salicyluric acids, from whole blood, isolated erythrocytes and plasma. Methylene chloride proved to be the best of the organic solvents tested. For whole blood and isolated erythrocytes it was necessary to carry out haemolysis prior to their extraction. The high-performance liquid chromatographic conditions for the quantitation of acetylsalicylic acid and its metabolites from samples of whole blood, erythrocytes and whole plasma were optimized. Separation was performed using reversed-phase chromatography on Separon SGX C18 and ultraviolet detection at 236 nm. A mixture of methanol-water (80:100, v/v) was the mobile phase, acidified with perchloric acid to pH 2.5.