IN VITRO MEASUREMENT OF TOTAL ANTIOXIDANT CAPACITY OF CRATAEGUS MACRACANTHA LODD LEAVES.

UNLABELLED: Crataegus macracantha Lodd, family Rosaceae, is a very rare species in Europe, and unlike Crataegus monogyna is less investigated for pharmacologic activity. AIM: To analyze the ability of the lyophilisate of extract obtained from leaves of Crataegus macracantha Lodd (single plant at the Iasi Botanical Garden) to capture free radicals in vitro. MATERIAL AND METHODS: The lyophilisate obtained in Department of Pharmacognosy, Faculty of Pharmacy, "Grigore T. Popa" University of Medicine and Pharmacy Iasi. The decreased absorbance of chromophore chlorpromazine radical cation in the presence of the lyophilized solutions was studied spectrophotometrically. The indicator radical cation, obtained by oxidation of chlorpromazine by potassium persulfate, has the maximum absorbance at 525 nm. Ascorbic acid was used as a standard antioxidant. RESULTS: The absorbance of radical solution was determined after the addition of a certain amount of lyophilisate at different time intervals. The antioxidant activity was calculated using the calibration curve obtained by plotting the variation in radical solution absorbance depending on ascorbic acid concentration. For each ascorbic acid concentration the area under the curve was calculated from plotting the percentage inhibition of the absorbance at two pre-established time intervals. CONCLUSIONS: The results confirm the antioxidant activity of the leaves of Crataegus Macracantha Lodd and by optimizing the proposed analytical methods the antiradical activity can be quickly evaluated with minimal reagent consumption.

Contributions to the phytochemical study of Bidens tripartitae herba from Romania. I. Tannins.

AIM: To analyze qualitatively and quantitatively tannins in the native plant, collected during the whole vegetation period from different areas of the country, and in its different organs (flower, stem, leaf). MATERIAL AND METHODS: For quantitative analysis, the plant product was extracted by repeated maceration (3 days) with 80% methanol. Proanthocyanidins in the extract were quantified by spectrophotometric methods. RESULTS: condensed tannins were present while hydrolyzed tannins were absent. Chromatographic analysis showed that tannins spectrum is similar in all plant organs and in plants collected at different times throughout the vegetation period. The differences are only quantitative. The maximum amount of tannins was found during the flowering stage (10.32%). CONCLUSIONS: In terms of tannin content, flowering is the best time to collect. However, collection throughout the whole vegetation period is acceptable.

[Development and validation of a GC-MS method for determination of nicotine in tobacco]. / Dezvoltarea si validarea unei metode GC-MS pentru determinarea nicotinei din tutun.

UNLABELLED: DETERMINATION FROM TOBA gas chromatographic-mass spectrometric method (GC/MS) for determination of nicotine from tobacco products was developed and validated. MATERIAL AND METHOD: It was used an Agilent Technologies 7890A Chromatograph equipped with a DB 5 MS column (30 m x 0.25 mm, 0.25 microm) and Agilent Technologies 5975C inert MSD detector. The temperature program began from 100 degrees C and increase with 10 degrees C/min to 190 degrees C than with 20 degrees C/min to 280 degrees C, constant for 5 min, with He as carrier gas (1 ml/min) and MS detection. The temperature of the source was 230 degrees C and the temperature of the quadrupole was 150 degrees C. The parameters of validation were studied according to the international requirements. RESULTS: Finally, the method established has following parameters: is linear in the 1.01-201.8 microg/ml range, has the detection limit of 3.6 microg/ml, the quantification limit of 10.8 microg/ml, is precise (RSD < 5%, n = 9) and accurate (mean recovery is 100.28% in 99.08-100.96 microg/ml range). CONCLUSION: The method can be used to determine nicotine from tobacco products, with good results.

Spectrophotometric determination of enalapril through ternary complex formation.

UNLABELLED: Enalapril is an angiotensin-converting enzyme inhibitor widely used in the treatment of hypertension and heart failure. AIM: To design and validate a simple and sensitive spectrophotometric method. This method is based on a ternary complex formation between enalapril, copper (II), and bromothymol blue, in a slightly alkaline environment, complex that is extractable in chloroform, with an absorption maximum at 426 nm. MATERIALS AND METHODS: To a volume of 1 milliliter of standard enalapril drug solution in concentration range 200/500 microg/ml, two milliliters of Cu (II) chloride solution 0.2 % followed by 2 ml bromothymol blue solution 0.01% and 0.5 ml of pH 8 buffer solution (Britton-Robinson) were added. The complex was two times extracted with 2 x 1.5 ml chloroform. Ten minutes after extraction, absorbance was measured at 426 nm against blank. RESULTS: Our spectrophotometric method was validated by determining linearity, limit of detection and quantification, system and method precision, and method accuracy. The method showed a good linearity in the range 200/500 microg/ml (correlation coefficient r = 0.9993). Limit of detection (LD) was 9.907 microg/ml and limit of quantification (LQ) 33.024 microg/ml. Precision showed an RSD = 1.83%, and accuracy with mean recovery is 102.6% in the range 98.8/103.9. CONCLUSIONS: The ternary complex formed under the above-mentioned condition and measured spectrophotometrically can be regarded as an ion-association complex between the metal-drug cation and bromothymol blue anion. The experimental results demonstrated a good sensitivity.

[Validation of a HPLC method for ochratoxin A determination]. / Validarea unei metode HPLC pentru determinarea ochratoxinei A.

Ochratoxin A is a mycotoxin produced by various species of Aspergillus and Penicillium. Ochratoxin A has been detected in cereals and cereal products, coffee beans, beer, wine, spices, pig's kidney and cow's milk. For ochratoxin A, a HPLC method was developed and validated. Ochratoxin A was determined by RP-HPLC, using a liquid chromatograph type HP 1090 Series II, equiped with a fluorescence detector. The analysis was performed with a Phenomenex column, type Luna C18(2) 100A (150 x 4.6 mm; 5 microm) with a mobile phase consisting of a mixture of acetonitrile/water/acid acetic (99/99/2), a flow of 0.7 mL/min. For detection, the wavelenght of excitation was 228 nm and wavelenght of emision was 423 nm. The calibration graph was linear in 6.25-50 ng/mL concentration range (r2 = 0,9991). The detection limits was 1.6 ng/mL and the quantification limit was 4.9 ng/mL. The method precision (RSD = 2.4975%) and the accuracy (recovery was 100.1%) were studied. The HPLC method was applyed for ochratoxin A from food samples with good results.

[Contribution to the mobile phase selection for HPLC separation in order to make the identification and quantitative determination using the UV-VIS detector]. / Contributii la alegerea fazei mobile in vederea separarii prin HPLC si identificarea--determinarea cantitativa a unor antiinflamatoare nesteroidiene folosind detectorul UV-VIS.

UNLABELLED: The aim of this study is to elaborate a HPLC with UV detection method for the analysis of some AINS, method which can be transferred in a system with mass spectrometry detection. MATERIAL AND METHODS: More AINS drugs (diclofenac sodium, ibuprophen, ketoprofen, meloxicame, piroxicame, tenoxicame, nimesulide, phenylbutazone and indomethacin) were studied by UV spectrophotometry and by HPLC. RESULTS: The UV spectra of the studied AINS were recorded in pure solvents or in a mixture of solvents in order to establish the absorbance that will be used for HPLC detection in UV. To establish the optimum chromatographic parameters, the influence of the flow and of the composition of the mobile phase (both of the aqueous and organic phases) were studied too. CONCLUSIONS: The proposed procedure is easy and relatively with low cost regarding the MS detection.

[Determination of methanol concentration by GC-MS in a fatal case poisoning]. / Determinarea concentratiei metanolului prin GC-MS într-un caz de intoxicatie letala.

UNLABELLED: Due to the consumption of alcoholic beverages with methanol content above the admitted limit, a woman from Bacau died at the Bacau County Hospital after 9 hours of hospitalization. MATERIALS AND METHODS: For the toxicological examination blood, liver, bile, stomach, intestine and kidney samples were analyzed in order to establish the concentration of methanol. Determination of methanol was carried out by gas chromatography coupled with mass spectrometry (GC-MS), analyzing the distillates obtained from blood and organs. We used an Agilent Technologies 7890 A gas chromatograph coupled to an Agilent Technologies 7683 C spectrometer. Helium was employed as mobile phase and a Phenomenex Zebron ZW-Plus polar column represented the stationary phase. Spectra acquisition mode: SIM, mass range followed: 15-500 atomic mass units, interpretation of the obtained results: Agilent Technologies ChemStation software. RESULTS: After peak integration on the chromatograms, the following values of methanol concentration were obtained: 1.720 g per thousand in blood, 0.795 g per thousand in liver, 2.661 g per thousand in bile, 2.287 g per thousand in stomach, 1.639 g per thousand in intestine and 1.590 g per thousand in kidney; the results have proved a large quantity of ingested methanol. They have also highlighted the absorption and distribution pattern of the methanol within the body.

[Construction and characterization of a selective membrane electrode for tenoxicam determination]. / Constructia si caracterizarea electrodului membrana selectiv pentru determinarea tenoxicamului.

UNLABELLED: This paper describes the construction and characterization of a selective membrane electrode which can be used for determination of tenoxicam. MATERIAL AND METHOD: The electroactive compound is a precipitate obtained in 2 N hydrocloric acid solution containing tenoxicam in which a solution of iodine is added. The membrane is made by mixing the electroactive compound with polyethylene using tetrahydrofurane as solvent. The solution is evaporated in order to obtain a thick membrane, which is attached at one end of a PVC tube and is fixed with the same polymeric solution. In this tube an internal Ag/AgCl reference electrode is inserted. The assembly is filled with an internal solution containing tenoxicam. The electrode was characterized (electrode slope, selectivity, optimal pH range, response time, life time). The developed method was validated. RESULTS: The method showed a good liniarity in the range of 10(-6)-10(-1) M (the correlation coefficient r = 0.9999). The detection limit (LD) was 7.347 x 10(-7) M and the quantification limit (LQ) was 1.017 x 10(-6) M. There were established the precision (RSD = 1.79%) and the accuracy (mean recovery is 100.17%) CONCLUSIONS: The experimental results demonstrated a good sensibility.

[Comparative data regarding two HPLC methods for determination of isoniazid]. / Date comparative privind doua metode de determinare prin HPLC a isoniazidei.

UNLABELLED: For the determination of isoniazide (isonicotinic acid hydrazide - HIN) two different HPLC methods were developed and validated. METHODS: Both experiments were performed using a Waters 2695 liquid chromatograph and a UV - Waters 2489 detector. The first method (I) used a Nucleosil 100-10 C18 column (250 x 4.6 mm), a mobile phase formed by a mixture of acetonitrile/10(-2) M oxalic acid (80/20) and a flow of 1.5 mL/ min; detection was done at 230 nm. The second method (II) used a Luna 100-5 C18 column (250 x 4.6 mm), a mobile phase formed by a mixture of methanol/acetate buffer, pH = 5.0 (20/ 80), a flow of 1 mL/min; detection was done at 270 nm. RESULTS: Both methods were validated, the correlation coefficients were 0.9998 (I) and 0.9999 (II), the detection limits were 0.6 microg/mL (I) and 0.055 microg/mL (II), the quantitation limits were 1.9 microg/mL (I) and 0.2 microg/ mL (II). There were also studied: the system precision (RSD = 0.1692% (I) and 0.2000% (II)), the method precision (RSD = 1.1844% (I) and 0.6170% (II)) and the intermediate precision (RSD = 1.8058% (I) and 0.5970% (II)). The accuracy was good, the calculated recoveries were 102.66% (I) and 101.36 (II). CONCLUSIONS: Both validated methods were applied for HIN determination from tablets with good and comparable results.

[Spectrophotometric determination of piroxicam using ferric ferricyanide as reagent]. / Determinarea spectrofotometrica în vizibil a piroxicamului folosind ca reactiv fericianura ferica.

UNLABELLED: For the piroxicam determination (nonsteroidal antiinflammatory drug-NSAID) it was developed a spectrophotometric method, based on the reduction of ferric ferricyanide into ferro-ferricyanide (Prussian Blue), with maximum of absorbance at 760 nm. MATERIAL AND METHOD: The practical working conditions were established. In the 0.2 divided by 2.0 microg/mL range of piroxicam concentration, were used the 2 mL of ferric ferricyanide 1 mL of 2N hydrocloric acid. To delay the flocculation of Prussian Blue it was to add a 1 mL solution of sodium lauryl sulfate 1%. After 15 minutes read the absorbance at 760 nm. The developed method was validated. RESULTS: The method showed a good linearity in the range of 0.2 divided by 2.0 microg/mL (the correlation coefficient r = 0.9995). The detection limit (LD) was 0.056 microg/mL and the quantification limit (LQ) was 0.18 microg/mL. There were established the system precision (RSD = 0.25%), the precison (RSD = 1.91%) and the accuracy-recovery in the range 98.21 divided by 104.92% with a mean recovery of 100.91%. CONCLUSION: The experimental results demonstrated a good sensibility. The specific absorptivity for this method is A(1 cm, 760 nm)(1%) = 4374 much higher than piroxicam in UV (A(1 cm, 330 nm)(1%) = 296)

[Ultraviolet spectrophotometric determination of tenoxicam using iodine solution as reagent]. / Determinarea spectrofotometrica in ultraviolet a tenoxicamului folosind ca reactiv solutia de iod.

UNLABELLED: For the tenoxicam determination (Nonsteroidal Antiinflammatory Drug - NSAID) it was developed a spectrophotometric method, after the coupling reaction of tenoxicam with iodine, in methanolic medium, with maximum of absorbance at 289 nm. MATERIALS AND METHOD: The practical working conditions were established. In the 0.5 divided by 5.0 microg/mL range of tenoxicam concentration, were used 5 x 10(-3) M iodine solution and 0.5 N hydrocloric acid. The stability of product were evaluated for 30 minutes. The developed method was validated. RESULTS: The method showed a good linearity in the range of 0.5 divided 5.0 microg/mL (the correlation coefficient r = 0.9995). The detection limit (LD) was 0.14 microg/mL and the quantification limit (LQ) was 0.49 microg/mL. There were established the precison (RSD = 1.90%) and the accuracy-recovery in the range 97.27 divided by 102.56% with a mean recovery of 99.49%. CONCLUSIONS: The experimental results demonstrated a good sensibility. The specific absorptivity for this method is A1%(1 cm,289 nm) = 1770 much higher than tenoxicam in methanol (A1%(1 cm,360 nm) = 323).

[Nimesulide spectrophotometric determination in the visible region]. / Determinarea spectrofotometrica in vizibil a nimesulidului.

UNLABELLED: Nimesulide is a nonsteroidal antiinflammatory (NSAID) drug whose mechanism of action is characterized by selective inhibition of cyclooxygenase-2. Nimesulide have analgesic and antipyretic properties. MATERIALS AND METHOD: For nimesulide analysis we developed a spectrophotometric method based on reduction of the nitro group of nimesulide by zinc and hydrochloric acid followed by diazotization and coupling with thymol (5-methyl-2-(1-methylethyl) phenol) in ammonia medium to form a stable chromophore which absorbs at 476 nm. There were established the practical working conditions. The developed method was validated. RESULTS: The method showed a good linearity in the range of 5 - 40 microg / sample (the correlation coefficient r = 0.9990). The detection limit (LD) was 0.99 microg/sample and the quantification limit (LQ) was 3.32 microg/sample. There were established the system precision (RSD = 1.70%), the precision (RSD < 2.2%) and the accuracy (recovery in the range 97.66 - 102.69% with a mean recovery of 99.81%) of the method. CONCLUSIONS: A new method for nimesulide spectrophotometric quantification in visible has been developed.

Prolonged gastroprotective activity of a ranitidine-dextran conjugate.

The paper presents the influence of the ranitidine-dextran conjugate on the gastric secretion stimulated with carbachol on rats with a ligated pylorus [correction of ligaturated pilor]. The gastric lesions, the gastric juice volume and the total acidity at 6, 24 and 48 hours after the treatment were examined. Ranitidine serum content was determined by HPLC. Administration of the ranitidine-dextran conjugate produces a higher inhibition of gastric lesions at 48 hours than the administration of the free drug (61.4% versus 33.9%) and a prolonged action for more 48 hours. Synthesis of a macromolecular prodrug of ranitidine and its in vitro behavior was reported in a previous paper (11). The present paper studies the performances obtained in gastro-protective action by using the new ranitidine-dextran conjugate reported (11).