Diagnostic Accuracy of Drop Hydrogen Peroxide Test as a Novel Bedside Diagnostic Test to Differentiate Transudative and Exudative Pleural Effusion Against Light's Criteria.

Background Diagnostic evaluation of pleural fluid according to Light's criteria to differentiate between exudative and transudative fluid takes 1 or 2 working days. For rapid clinical management, especially in critically ill patients, a simpler bedside diagnostic test can be done which has similar diagnostic accuracy as that of Light's Criteria. Objective To determine the diagnostic accuracy of Drop Hydrogen Peroxide test to differentiate exudative and transudative pleural effusion in comparison to Light's criteria. Method A concurrent validity test was performed using a convenient sampling technique including patients presenting to the Department of Internal Medicine from January to September 2021, who had pleural effusion. Two milliliters of tapped pleural fluid of patients who underwent aseptic thoracocentesis was collected in a test tube to which one to two drops of 20% hydrogen peroxide was added. Presence of bubbles suggested an exudative type of fluid. Rest of the tapped pleural fluid was sent to the laboratory for further evaluation by Light's criteria, which was compared with the results by Drop Hydrogen Peroxide Test. Result There were 83 patients who had pleural effusion, of them a total of 43 patients had transudative pleural effusion while 40 patients had exudative pleural effusion based on Light's criteria and 37 patients had transudative pleural effusion while 46 patients had exudative pleural effusion based on drop hydrogen peroxide test. Conclusion The drop hydrogen peroxide test allows cost effective and prompt evaluation of the type of pleural effusion is exudative or transudative, thereby making it a convenient diagnostic bedside test.

Urinary B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL): potential biomarkers of active lupus nephritis.

B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) help in B cell activation, maintenance and plasma cell survival. B cell infiltration has been demonstrated in kidneys of patients with lupus nephritis (LN). Serum levels of BAFF and APRIL have shown inconsistent relationships with lupus disease activity. We evaluated urinary levels of BAFF and APRIL as biomarker for LN. Thirty-six patients with proliferative lupus nephritis (AN), 10 with active lupus without nephritis (AL) and 15 healthy controls (HC) were studied. APRIL and BAFF levels were measured in both serum and urine using enzyme-linked immunosorbent assay (ELISA). Urine levels were normalized for urinary creatinine excretion. Urine levels were correlated with conventional disease activity markers and histology. Levels were reassessed in 20 AN patients at 6 months after treatment with cyclophosphamide. Urinary APRIL (uAPRIL) and BAFF (uBAFF) levels were raised significantly in AN. uAPRIL, but not uBAFF, correlated moderately with renal Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) in AN (r = 0·36, P < 0·05). On receiver operator curve (ROC) analysis, uBAFF and uAPRIL showed an area under the curve (AUC) of 0·825 and 0·781, respectively, in differentiating between nephritis and non-nephritis, which performed better than low C3, C4 and raised anti-dsDNA antibodies. There was no correlation of serum levels with uBAFF (r = 0·187, P = 0·261) and uAPRIL (r = 0·114, P = 0·494). uAPRIL levels reduced after treatment (mean 125 pg/mg to 36 pg/mg, P < 0·05). uBAFF levels reduced in 16 responders while two of four non-responders had increase in levels. Thus, uBAFF and uAPRIL are potential biomarkers of proliferative lupus nephritis.

Recombinant Salmonella typhimurium outer membrane protein A is recognized by synovial fluid CD8 cells and stimulates synovial fluid mononuclear cells to produce interleukin (IL)-17/IL-23 in patients with reactive arthritis and undifferentiated spondyloarthropathy.

In developing countries, one-third of patients with reactive arthritis (ReA) and undifferentiated spondyloarthropathy (uSpA) are triggered by Salmonella typhimurium. Synovial fluid mononuclear cells (SFMCs) of patients with ReA and uSpA proliferate to low molecular weight fractions (lmwf) of outer membrane proteins (Omp) of S. typhimurium. To characterize further the immunity of Omp of Salmonella, cellular immune response to two recombinant proteins of lmwf, OmpA and OmpD of S. typhimurium (rOmpA/D-sal) was assessed in 30 patients with ReA/uSpA. Using flow cytometry, 17 of 30 patients' SF CD8(+) T cells showed significant intracellular interferon (IFN)-γ to Omp crude lysate of S. typhimurium. Of these 17, 11 showed significantly more CD8(+) CD69(+) IFN-γ T cells to rOmpA-sal, whereas only four showed reactivity to rOmpD-sal. The mean stimulation index was significantly greater in rOmpA-sal than rOmpD-sal [3·0 (1·5-6·5) versus 1·5 (1·0-2·75), P < 0·005]. Similarly, using enzyme-linked immunospot (ELISPOT) in these 17 patients, the mean spots of IFN-γ-producing SFMCs were significantly greater in rOmpA-sal than rOmpD-sal [44·9 (3·5-130·7) versus 19·25 (6-41), P < 0·05]. SFMCs stimulated by rOmpA-sal produced significantly more proinflammatory cytokines than rOmpD-sal: IFN-γ [1·44 (0·39-20·42) versus 0·72 (0·048-9·15) ng/ml, P < 0·05], interleukin (IL)-17 [28·60 (6·15-510·86) versus 11·84 (6·83-252·62) pg/ml, P < 0·05], IL-23 [70·19 (15-1161·16) versus 28·25 (> 15-241·52) pg/ml, P < 0·05] and IL-6 [59·78 (2·03-273·36) versus 10·17 (0·004-190·19) ng/ml, P < 0·05]. The rOmpA-sal-specific CD8(+) T cell response correlated with duration of current synovitis (r = 0·53, P < 0·05). Thus, OmpA of S. typhimurium is a target of SF CD8(+) T cells and drives SFMC to produce increased cytokines of the IL-17/IL-23 axis which contribute to the pathogenesis of Salmonella-triggered ReA.

Study of reaction parameters and kinetics of esterification of lauric acid with butanol by immobilized Candida antarctica lipase.

Esterification of lauric acid with n-butanol, catalyzed by immobilized Candida antarctica lipase (CAL) in aqueous-organic biphasic solvent system was studied. Effects of various reaction parameters on esterification were investigated, such as type and amount of solvent, amount of buffer, pH, temperature, speed of agitation, amount of enzyme, butanol and lauric acid. The most suitable reaction conditions for esterification were observed at 50 degrees C and pH 7.0 using 5000 micromoles of lauric acid, 7000 pmoles of butanol, 0.25 ml phosphate buffer, 1 ml of isooctane as the solvent and 50 mg of immobilized enzyme in the reaction medium at agitation speed of 150 rpm. Maximum esterification of 96.36% was acheived in 600 min of reaction time at n-butanol to lauric acid molar ratio of 1: 0.7. Kinetic study for the esterification of lauric acid with n-butanol using immobilized CAL was carried out and the kinetic constants were estimated by using non-linear regression method. The estimated value of Michaelis kinetic constants for butanol (KmBt) and acid (KmAc) were 451.56 (M) and 4.7 x 10(-7)(M), respectively and the value of dissociation constant (KBt) of the butanol-lipase complex was 9.41 x 10(7)(M). The estimated constants agreed fairly well with literature data.

A mouse model of lamellar intrastromal femtosecond laser keratotomy: ultra-structural, inflammatory, and wound healing responses.

PURPOSE: The availability of knockout mouse species provide a highly versatile platform for critically examining the corneal wound healing response. We aimed to develop and characterize the wound healing response in a mouse model of intrastromal femtosecond laser (FSL) keratotomy. METHODS: An intrastromal lamellar dissection using a Visumax FSL was performed on 16 wild type mice (C57BL6) . The energy level was optimized at 150nJ. The FSL was programmed to perform a lamellar dissection at 50 µM depth without sidecut. The flap was not lifted. Fellow eyes were used as controls. Slit lamp photography and confocal microscopy were performed immediately before the mice were sacrificed 4 h, 1, 3, and 7 days post surgery. Corneas were harvested for immunocytochemistry, transmission electron microscopy (TEM) and light microscopy (LM). RESULTS: Confocal microscopy showed an absence of keratocytes in the area immediately surrounding the dissection plane. The dissection plane and individual FSL plasma cavitation bubbles were clearly evident on TEM. There was evidence of Keratocyte cell death along the laser resection plane on TEM. LM revealed the dissection plane at a 20 µM depth, although not all epithelial cell layers were intact. Staining for monocytes using antibodies for CD11b (cluster of differentiation 11b) showed early migration at the peripheries at 4 h that increased at 24 h and became more central in treated corneas (p<0.001). Apoptotic cells were evident on TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay in the immediate ablation zone and were significantly raised at 4 and 24 h (p<0.001). Ki67 (Kiel 67 protein) positive proliferating keratocytes are evident at 3 days and increased significantly by 7 days (p<0.001). Minimal fibroblast (cluster of differentiation 90, CD90) transformation was seen at 1 week. No myofibroblasts were detected. DISCUSSION: We have demonstrated that FSL lamellar cuts can be effectively performed on mice and that this model exhibits typical signs of the corneal wound healing response. This model could provide a ubiquitous platform in which to study corneal wound healing responses in both wild type and knockout mice species. The ability to create such a lamellar pocket may be utilizzd for intrastromal drug delivery.

Stromal interleukin-1 expression in the cornea after haze-associated injury.

The purpose of this study was to determine whether myofibroblasts or other cells in the stroma in the cornea produce interleukin (IL)-1alpha or IL-1beta that could modulate myofibroblast viability in corneas with haze after photorefractive keratectomy (PRK). Twenty-four female rabbits had haze-generating PRK for 9 diopters of myopia and were sacrificed at 1 week, 2 weeks, 3 weeks or 4 weeks after surgery. Corneal rims were removed, frozen in OCT at -80 degrees C, and analyzed by immunocytochemistry using primary antibodies to IL-1alpha, IL-1beta and alpha smooth muscle actin (SMA). Double immunostaining was performed for the co-localization of SMA with IL-1alpha or IL-1beta. Central dense haze and peripheral slight haze regions of each cornea were analyzed. SMA+ cells that expressed IL-1alpha protein were detected in both regions of the corneas at most time points following PRK. However, in the haze region at the 1, 3 and 4 week time points, significantly more (p<0.01) SMA+ cells did not express IL-1alpha. Also, in the haze region at all three time points, significantly more (p<0.01) SMA- cells than SMA+ cells expressed interleukin-1alpha protein. IL-1beta expression patterns in SMA+ and SMA- stromal cells was similar to that of IL-1alpha after PRK. Previous studies have demonstrated that IL-1alpha or IL-1beta triggers myofibroblast apoptosis in vitro, depending on the available concentration of apoptosis-suppressive TGFbeta. This study demonstrates that SMA- cells such as corneal fibroblasts, keratocytes, or inflammatory cells may produce IL-1alpha and/or IL-1beta that could act in paracrine fashion to regulate myofibroblast apoptosis--especially in the region where there is haze in the cornea after PRK was performed and SMA+ myofibroblasts are present at higher density. However, some SMA+ myofibroblasts themselves produce IL-1alpha and/or IL-1beta, suggesting that myofibroblast viability could also be regulated via autocrine mechanisms.

Activity and stability of biochar in hydrogen peroxide based oxidation system for degradation of naphthenic acid.

This study investigated the stability and catalytic activity of wheat straw biochar (WS), hardwood biochar (HW) and commercial activated carbon (AC) in hydrogen peroxide (H2O2) based oxidation system for degradation of model naphthenic acids compound, 1-methyl-1- cyclohexane carboxylic acid (MCCA). WS showed excellent catalytic activity for decomposition of H2O2 and MCCA degradation as demonstrated by high H2O2 decomposition rate (2.0*10-4 M-1s-1), amount of hydroxyl (OH) radicals generated (182 mg/L) and degradation efficiency of MCCA (100% at Co - 100 mg/L). 2-Methyl pentatonic acid was identified as reaction intermediate and 99% mineralization of MCCA was obtained within 4 h. The real wastewater conditions were simulated by addition of chloride (Cl-) and bicarbonate ions (HCO3-) and found that lower concentrations of Cl- and HCO3- have minimal influence on MCCA removal. Overall, biochar catalyzed H2O2 based oxidation process has great potential and can be applied for degradation of NAs in oil-sand processed water.

Corneal thickness in uveal coloboma with microcornea.

PurposeTo evaluate the relationship between central corneal thickness (CCT) and horizontal corneal diameter (HCD) in eyes with uveal coloboma and microcornea.Patients and methodsAll adult patients with uveal coloboma having microcornea in at least one eye and seen between May 2014 and July 2016 at the cataract clinic of our tertiary eye care center were evaluated. The study group comprised 26 colobomatous eyes of 15 patients who had associated microcornea. The control group comprised eyes with uveal coloboma and normal-sized cornea (n=12 eyes of 9 patients). The main outcome measure included the relationship between CCT and HCD in eyes with uveal coloboma and microcornea.ResultsThe mean HCD of the study and control groups was 6.97±1.28 and 11.39±0.67 mm, respectively. The mean central pachymetry of the study group was 668.4±40.2 µm compared with 555.3±41.8 µm in the control group. In both the groups, CCT was negatively correlated with HCD (R=-0.62 in the study group and R=-0.72 in the control group).ConclusionsUveal colobomatous eyes with microcornea have thicker corneas compared with uveal colobomatous eyes with normal diameter corneas that have normal central corneal thickness. In all the eyes with uveal coloboma, there is a negative correlation between horizontal corneal diameter and central corneal thickness.

Determination of traces of chloride and fluoride in H2SO4, H3PO4 and H3BO3 by in situ analyte distillation-ion chromatography.

A simple dual vessel in situ analyte distillation (IAD) system has been developed for suppressed ion chromatographic determination of chloride and fluoride ions in complex matrices. In IAD system, water vapours generated from the outer vessel reacts with sulfuric acid generating heat, thus favouring the quantitative distillation of chloride and fluoride within 30 min on water bath temperature (approximately 80 degrees C). The distilled analytes, as their respective acids in water, were directly injected into an ion-chromatograph. This newly developed method has been applied for analysis of trace impurities in H2SO4, H3PO4 and H3BO3. The detection limits for chloride is 8, 80 and 70ppb (w/w) for H2SO4, H3PO4 and H3BO3, respectively. For fluoride the detection limits are 6 and 60 ppb (w/w) for H2SO4 and H3PO4, respectively. The recovery of spikes for both the analytes ranged between 87 and 100%.

Protective effects of vitamin E against lead-induced deterioration of membrane associated type-I iodothyronine 5'-monodeiodinase (5'D-I) activity in male mice.

The protective role of vitamin E (vit E) on lead-induced thyroid dysfunction with special reference to type-I iodothyronine 5'-monodeiodinase (5'D-I) activity in mice liver was investigated. Daily intraperitoneal (i.p.) injection of lead acetate (0.5 mg/kg body weight) for 30 days significantly decreased serum 3,3',5-triiodothyronine (T3) concentration and hepatic 5'D-I activity. Furthermore, lead significantly increased peroxidative reactions involving membrane components (lipid peroxidation, LPO) while the activities of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) were decreased in mouse liver. Simultaneous administration of vit E (5 mg/kg body weight) and 0.5 mg/kg body weight of lead restored thyroid function in mice by maintaining normal hepatic 5'D-I activity and serum thyroid hormone concentrations. It also prevented increase in LPO and inhibition of SOD and CAT activities in liver. We suggest that the intact membrane structure is a must for 5'D-I activity and the administration of vit E may prevent the lead induced thyroid dysfunction by maintaining membrane architecture.

Procedure for determination of trace ions in boric acid by matrix volatilization-ion chromatography.

A method for determination of anions and cations in boric acid is proposed by matrix volatilization. The boric acid matrix was eliminated as trimethyl borate ester in a vapour phase matrix elimination (VPME) system using a mixture of glycerol-methanol. In this VPME system, in situ reagent purification, sample decomposition and digest evaporation were achieved in a single step. Trace anions were separated on anion-exchange column (IonPac AS17) by an isocratic elution with 15 mM sodium hydroxide and the cations on a cation-exchange column (IonPac CS12) by 20 mM hydrochloric acid as eluents. Method detection limits (3sigma) for most ions ranged from 0.3 to 8 ng/g (ppb). Recovery experiments combined with comparison of data obtained by other methods were employed to verify the accuracy of the proposed method. Application of the method to determine trace levels of anions like acetate, oxalate, sulfate, phosphate and cations such as lithium, sodium, potassium, magnesium and calcium in two highly pure grades of boric acid using ion chromatography is demonstrated.

In situ matrix evaporation by isothermal distillation of high-purity reagents for the determination of trace impurities by ion chromatography.

In situ matrix evaporation of high-purity acids based on isothermal distillation was achieved in a high-density polyethylene (HDPE) container on a water bath, to avoid contamination from the laboratory environment. The solubility of water and acid vapours in glycerol due to co-association was utilized to achieve complete evaporation. All major sources which contribute to the process blank were taken care of in a simple and effective way. A 50-fold preconcentration with >99.9% matrix removal was achieved for the analysis of low-boiling acids, HCl, HF, HNO3 and H2O2. The non-volatile ions NH4+, Li+, Na+, K+, Mg2+, Ca2+, SO4(2-) and PO4(3-) were determined by ion chromatograph with conductivity detection. The detection limits were 6-130 ng/l with recoveries of 85-110% for all ions studied.

Ion chromatographic determination of trace level phosphorus in purified quartz.

Trace levels of phosphorus in purified quartz are determined by ion chromatography. In situ reagent purification, matrix digestion and oxidation of phosphorus to orthophosphate ion are carried out simultaneously in a vapour phase digestion (VPD) assembly using a mixture of HF, HNO3 and H2O2. A drastic reduction (475 times) in phosphate blank from reagents (HF/H2O2) was achieved in the VPD through in situ purification of the reagent. The residues remaining after volatilisation (solvent/matrix), mostly consisting of insoluble phosphate/fluoride salts of divalent and trivalent cations, were solubilised by ion-exchange dissolution. Phosphate was analysed on the IonPac AS17 column with suppressed conductivity detection. The results of the ion chromatography (IC) method were compared with a spectrophotometric method. Accuracy was evaluated by analysing a certified reference material (silicon, NIST 57a). The method detection limit was 0.05 microg g(-1).

Industrial effluent B.O.D. Quanta and expected causes.

The biological oxygen demand (B.O.D.) is an important parameter for the measurement of organic pollutants in waste water. The present paper deals with the various causes which may be responsible for B.O.D. variations in waste water of different types of industrial effluents.

Antioxidant and anti-inflammatory property of Sandhika: a compound herbal drug.

The present study was envisaged to assess the rationality for the use of "Sandhika", a popular Ayurvedic drug in rheumatoid arthritis. This drug, when tested against carrageenan induced paw oedema and cotton pellet granuloma, showed significant anti-inflammatory activity at the dose of 0.25 g/kg body weight. The antioxidant property was assessed by determining cumene hydroperoxide (CHP) induced lipid peroxidation and reduced glutathione content in rat liver homogenate (in vitro). Experiments show the significant protection against lipid peroxidation at the dose of 80 micrograms/ml, measured as reduction in the level of malondialdehyde (MDA) induced by 1.5 mM cumene hydroperoxide (CHP). This effect was accompanied by the maintained reduced glutathione (GSH) content in drug treated rats. Oral treatment of drug up to 2 g/kg body weight for 15 days did not show any rise in serum transaminases (SGOT and SGPT). The results suggest that "Sandhika" which is an indigenous drug for inflammation with no detectable adverse effect, might be acting through scavenging the free radicals.

Bacopa monniera Linn. as an antioxidant: mechanism of action.

Bacopa monniera, Linn. (Brahmi: Scrophulariaceae) an Ayurvedic medicine is clinically used for memory enhancing, epilepsy, insomnia and as mild sedative. For the first time the effect of alcohol and hexane fraction of Brahmi has been studied on FeSO4 and cumene hydroperoxide induced lipid peroxidation. Alcohol fraction showed greater protection with both inducers. Results were compared with known antioxidants tris, EDTA and a natural-antioxidant vitamin E. The effect of Brahmi was also examined on hepatic glutathione content. The mechanism of action could be through metal chelation at the initiation level and also as chain breaker. The results suggested that Brahmi is a potent antioxidant. The response of Brahmi was dose dependent. Tris, an hydroxyl trapper did not show any protection in comparison to Brahmi where as EDTA and vitamin E did protect against FeSO4. In experimental conditions 100 micrograms Brahmi extract (alcoholic) was equivalent to 247 micrograms of EDTA (0.66 microM) and 58 micrograms of vitamin E. Interestingly Brahmi only slightly protected the autooxidation and FeSO4 induced oxidation of reduced glutathione on lower doses 100 micrograms/ml and below, but on higher concentrations it enhanced the rate of oxidation.

Unusual extensive physiologic melanin pigmentation of the oral cavity: a clinical presentation.

Pigmented lesions are commonly found in the oral cavity. Oral pigmentations may be physiological or pathological in nature. It may represent as a localized anomaly of limited significance or the presentation of potentially life threatening multisystem disease. Oral pigmentation has a multifactorial etiology. Most of the oral pigmentations are physiologic. Evaluation of a patient with pigmented lesions should include a full medical and dental history, extraoral and intraoral examinations. In this article, we report a case of extensive physiologic pigmentation of the oral cavity in a 12 year old female patient, posing a diagnostic challenge.

Thermal stability, complexing behavior, and ionic transport of polymeric gel membranes based on polymer PVdF-HFP and ionic liquid, [BMIM][BF4].

PVdF-HFP + IL(1-butyl-3-methylimidazolium tetrafluoroborate; [BMIM][BF(4)]) polymeric gel membranes containing different amounts of ionic liquid have been synthesized and characterized by X-ray diffraction, scanning electron microscopy, Fourier transform infrared (FTIR), differential scanning calorimetry, thermogravimetric analysis (TGA), and complex impedance spectroscopic techniques. Incorporation of IL in PVdF-HFP polymer changes different physicochemical properties such as melting temperature (T(m)), thermal stability, structural morphology, amorphicity, and ionic transport. It is shown by FTIR, TGA (also first derivative of TGA, "DTGA") that IL partly complexes with the polymer PVdF-HFP and partly remains dispersed in the matrix. The ionic conductivity of polymeric gel membranes has been found to increase with increasing concentration of IL and attains a maximum value of 1.6 × 10(-2) S·cm(-1) for polymer gel membrane containing 90 wt % IL at room temperature. Interestingly, the values of conductivity of membranes with 80 and 90 wt % of IL were higher than that of pure IL (100 wt %). The polymer chain breathing model has been suggested to explain it. The variation of ionic conductivity with temperature of these gel polymeric membranes follows Arrhenius type thermally activated behavior.

Endothelial keratoplasty in the management of irido-corneal endothelial syndrome.

PURPOSE: To describe the results of endothelial keratoplasty (EK) in the management of irido-corneal endothelial (ICE) syndrome. DESIGN: Retrospective, consecutive, interventional case series. METHODS: Eight eyes of seven patients underwent EK for corneal edema owing to ICE syndrome. Simultaneous cataract surgery was performed in five eyes. The charts were reviewed for demographics, clinical findings, and outcomes after EK. RESULTS: Mean age was 50.4 years (range 35-59 years). Mean follow-up was 12.5 months (range 6-24 months). Corneal clarity was restored in all eyes and was maintained till the last follow-up. Four patients had acute rise in the intraocular pressure in the post-operative period, which was well controlled on medical treatment. One patient who was non-compliant with medications and regular follow-up advice had progression of glaucomatous disc changes and visual field loss. The best-corrected visual acuity ranged from 20/50-20/30. CONCLUSIONS: EK is a viable option in ICE syndrome. Adequate control of glaucoma is the paramount factor in maintaining good visual acuity in these eyes.

Dry ashing of organic rich matrices with palladium for the determination of arsenic using inductively coupled plasma-mass spectrometry.

A dry ashing procedure is developed for the determination of As in organic rich matrices such as wheat flour, lichen and tobacco leaves. The volatility of As during dry ashing is avoided by the addition of palladium nitrate [Pd(NO(3))(2)]. The recovery of both As(III) and As(V) is found to be near quantitative. The residue after dry ashing is dissolved in nitric acid (HNO(3)) and analysed by inductively coupled plasma-mass spectrometry (ICP-MS). The process blank and limit of detection (LOD) are 11 and 6.6 ng g(-1), respectively. The procedure is applied for the determination of As in certified reference materials namely wheat flour NIST SRM 1567a (National Institute of Standards and Technology Standard Reference Material), lichen BCR CRM 482 (Institute for Reference Materials, European Commission) and Virginia tobacco leaves CTA-VTL-2 (Poland Academy of Sciences). The results obtained by the present procedure are in good agreement with the certified values and also determined after complete dissolution of samples using closed microwave digestion.