High-pressure Liquid Chromatographic Procedure Research Articles

A Reverse Phase Stability Indicating HPLC Method Development for Estimation Assay of Benzyl Alcohol and Glycopyrrolate in Glycopyrrolate Injection

Background: The present work is focused on developing a simple stability indicating method for the estimation of the assay of benzyl alcohol and glycopyrrolate in glycopyrrolate injection using reverse high performance liquid chromatography. Methods: A simple, selective, rapid, precise, and gradient reversed-phase high-pressure liquid chromatography procedure has been proposed to estimate the assay of benzyl alcohol and glycopyrrolate for glycopyrrolate injection. It was developed on a Kromasil 100-5, C8 250 mm x 4.6 mm, 5 μm column using mobile phase-A, containing pH 2.5 potassium dihydrogen phosphate buffer and methanol in a ratio of 80:20 v/v. and mobile phase-B, containing pH 2.5 potassium dihydrogen phosphate buffer and methanol in a ratio of 10:90 v/v at a flow rate of 1.5 mL/min. The detection of glycopyrrolate and benzyl alcohol was carried out at 222 nm and 254 nm. A sharp peak was obtained for glycopyrrolate and benzyl alcohol at a retention time of about 8.4 and 4.8 min. Results: The method was validated for specificity (blank, system suitability solution, standard, and sample and stress samples for any interference), precision (six replicate sample preparations), robustness (by slightly varying critical method parameters), and solution stability, and the results were found satisfactory. Conclusion: The method is specific, precise, accurate, and robust for estimation of the assay of benzyl alcohol of glycopyrrolate in glycopyrrolate injection.

Measurement of HbA1c in Gingival Crevicular Blood Using a High-Pressure Liquid Chromatography Procedure.

To validate an ion exchange high-pressure liquid chromatography (HPLC) method for measuring glycated hemoglobin (HbA1c) in gingival crevicular blood (GCB) spotted on filter paper, for use in screening dental patients for diabetes. We collected the GCB specimens for this study from the oral cavities of patients during dental visits, using rigorous strategies to obtain GCB that was as free of debris as possible. The analytical performance of the HPLC method was determined by measuring the precision, linearity, carryover, stability of HbA1c in GCB, and correlation of HbA1c results in GCB specimens with finger-stick blood (FSB) specimens spotted on filter paper. The coefficients of variation (CVs) for the inter- and intrarun precision of the method were less than 2.0%. Linearity ranged between 4.2% and 12.4%; carryover was less than 2.0%, and the stability of the specimen was 6 days at 4°C and as many as 14 days at -70°C. Linear regression analysis comparing the HbA1c results in GCB with FSB yielded a correlation coefficient of 0.993, a slope of 0.981, and an intercept of 0.13. The Bland-Altman plot showed no difference in the HbA1c results from the GCB and FSB specimens at normal, prediabetes, and diabetes HbA1c levels. We validated an HPLC method for measuring HbA1c in GCB; this method can be used to screen dental patients for diabetes.

Advanced ultra high pressure liquid chromatography–tandem mass spectrometric methods for the screening of red wine anthocyanins and derived pigments

Anthocyanins are responsible for the colour of red grapes and wine. In addition to their contribution to the sensory properties of wine, these compounds are also of interest due to their beneficial biological properties. Wine anthocyanins exhibit a large structural diversity due to variations in glycosylation and acylation patterns, which is further exacerbated by the diverse reactions involving grape-derived anthocyanins during wine ageing. Chromatographic as well as mass spectrometric resolution of wine anthocyanins is often precluded due to the complexity of these compounds. In this paper we report a rapid, high-efficiency ultra high pressure liquid chromatography (UHPLC) procedure with tandem mass spectrometric (MS/MS) detection for the in-depth screening of wine pigments. Selective detection of wine anthocyanins and derived pigments was achieved utilizing MS/MS in neutral loss scanning mode to observe the loss of dehydrated sugar moieties. This facilitated tentative compound identification based on molar mass information as well as the structured elution order of these compounds. In a second experiment, product ion spectra were recorded to allow identification of the anthocyanidin base using characteristic fragmentation patterns. The proposed methodology therefore involves two analyses for the sensitive and accurate identification of anthocyanins and their derived products in red wines. Mass spectra of wine anthocyanins under high energy collision induced dissociation (CID) conditions are reported, some for the first time. Significantly, chemical alteration of anthocyanins during wine ageing results in an off-set of the predominant fragments for each anthocyanidin base, whilst maintaining similar relative intensities. This allows unambiguous assignment of the derived products of anthocyanidin-glycosides. Using this approach, a total of 121 anthocyanins and derived compounds were identified in wines based on their relative reversed phase elution order as well as mass spectral information.

Taxonomic study and partial characterization of antimicrobial compounds from a moderately halophilic strain of the genus Actinoalloteichus.

A moderately halophilic actinomycete strain designated AH97 was isolated from a saline Saharan soil, and selected for its antimicrobial activities against bacteria and fungi. The AH97 strain was identified by morphological, chemotaxonomic and phylogenetic analyses to the genus Actinoalloteichus. Analysis of the 16S rDNA sequence of strain AH97 showed a similarity level ranging between 95.8% and 98.4% within Actinoalloteichus species, with A. hymeniacidonis the most closely related. The comparison of the physiological characteristics of AH97 with those of known species of Actinoalloteichus showed significant differences. Strain AH97 showed an antibacterial and antifungal activity against broad spectrum of microorganisms known to be human and plant pathogens. The bioactive compounds were extracted from the filtrate culture with n-butanol and purified using thin layer chromatography and high pressure liquid chromatography procedures. Two active products were isolated, one hydrophilic fraction (F1) and another hydrophobic (F2). Ultraviolet-visible, infrared, mass and 1H and 13C nuclear magnetic resonance spectroscopy studies suggested that these molecules were the dioctyl phthalate (F2) and an aminoglycosidic compound (F1).

Biflavones and triterpenoids isolated from Ouratea castaneifolia (DC.) Engl., Ochnaceae

This paper presents the chemical investigation of the leaves and stems of Ouratea castaneifolia (DC.) Engl.. There are no chemical or pharmacological studies with this species. Classic phytochemical investigation of the organic extracts together with high pressure liquid chromatography (HPLC) procedures lead to the identification of seventeen metabolites: seven triterpenes (friedelin, 3β-friedelinol, α-amyrin, β-amyrin, lupeol, germanicol and taraxerol), four steroids (sitosterol, stigmasterol and the glycosides sitosteryl 3-O-β-D-glucopyranoside and stigmasteryl 3-O-β-D-glucopyranoside), one isoflavone (5,7,4'-trimethoxyisoflavone), one flavone (5,4'-dihydroxy-7,3',5'-trimethoxyflavone) and four biflavones (amenthoflavone, 7,7"-O-dimethylamenthoflavone, heveaflavone and tetramethylamenthoflavone). The structures of the compounds were established by the analysis of ¹H, 13C NMR spectra including bidimensional techniques. The classes of the identified metabolites are in agreement with previous studies of the Ouratea genus.

Flow Injection Spectrophotometric Determination of the Antibacterial Levofloxacin in Tablets and Human Urine

A sensitive and fast flow‐injection (FI) spectrophotometric method for the determination of levofloxacin based on the formation of a colored product upon oxidation with N‐bromosuccinimide (NBS) in acidic medium is proposed. Optimization of chemical and FI variables has been made. Under the optimized conditions, the sampling rate was over 90 h−1, the calibration curve obtained was linear over the range 10–300 µg·mL−1, and the detection limit was 3 µg·mL−1. The proposed method was successfully applied to the determination of levofloxacin in pharmaceuticals and human urine samples. It was also found that the excipients in the commercial tablet preparation did not interfere with the assay. Results are precise (RSD<2.7%; n =10) and in agreement with those found by the reference high pressure liquid chromatography (HPLC) procedure.

(18) Biodiversity of the Flavonoid Phlorizin in a Subset of the USDA Apple Germplasm Core Collection

Phlorizin is known for its role in reducing glucotoxicity and has a long history of use in diabetes research. In addition, its contribution to the pool of total phenolics adds to the overall health benefits attributed to fruit. Phlorizin is limited to Rosaceae family plants, of which apple comprises its current commercial source; however, limited information exists regarding its biodiversity among apple taxa. A subset of 22 taxa from a core collection of apple accessions representative of the global genetic diversity of apple was used to investigate the biodiversity of phlorizin present in apple shoots and in fruit relative to total phenolic content and free radical scavenging capacity. Fruit and shoots were harvested from the USDA Plant Genetic Resources Unit in Geneva, N.Y. Validation and quantification of phlorizin was conducted using a rigorous high-pressure liquid chromatography (HPLC) procedure. Total phenolics in fruit, assayed using a Folin-Ciocalteu method and expressed as gallic acid equivalents, ranged from 227 to 7181 mg·L-1 and were strongly related to 2,2' azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) antioxidant capacity for the core collection (r= 0.778). On a molar basis, phlorizin had lower antioxidant capacity than other major phenolic compounds present in apple fruit, but was more effective than ascorbic acid. Phlorizin yield in dormant apple shoots, expressed as percent weight, ranged from 0.9% to 5.5%. A rapid, 96 well micro-plate spectrophotometric assay was also developed to aid in the screening of multiple samples for selection of high phlorizin yielding apple taxa. Spectrophotometry overestimated phlorizin content as expected, but the calibration curve between HPLC and spectrophotometry was acceptable, r2 = 0.88.

RNA synthesis using 2'-O-(tert-butyldimethylsilyl) protection.

This chapter enables the reader to carry out the solid-phase synthesis of ribonucleic acid (RNA) using beta-cyanoethyl phosphoramidite chemistry combined with tert-butyldimethylsilyl protection of the ribose 2'-hydroxyl group. Phosphoramidite monomers are activated with 5-benzylmercapto-1H-tetrazole enabling fast and highly efficient coupling to the 5'-hydroxyl group of the support-bound oligonucleotide. On completion of the synthesis, the stepwise deprotection of the nucleobase, phosphate, and ribose protecting groups is carried out using optimized protocols. Subsequently the various high-pressure (performance) liquid chromatography (HPLC) procedures are described enabling the purification and analysis of the RNA. For this purpose anion-exchange and reversed-phase HPLC are used singly or in combination according to the final purity requirement of the RNA.

Paralytic shellfish poisoning (PSP) in Margarita Island, Venezuela.

A severe outbreak of Paralytic Shellfish Poisoning (PSP) occurred in Manzanillo and Guayacán, northwestern coast of Margarita Island, Venezuela, between August and October 1991. A bloom of dinoflagellates including Prorocentrum gracile, Gymnodinium catenatum and Alexandrium tamarense seemed to be responsible for this outbreak. Levels of PSP toxins in mussels (Perna perna) exceeded the international safety limit of saxitoxin, 80 microg STX/100 microg meat. PSP toxin values varied between 2548 and 115 microg STX/100 g meat in Manzanillo, and between 1422 and 86 microg STX/100 g meat in Guayacán. At both locations, the highest levels were detected in August, when 24 patients exhibited typical symptoms of PSP toxicity after consuming cooked mussels (16 required hospitalization). A high pressure liquid chromatographic (HPLC) procedure was recently used on the 1991 samples. The major toxin detected in samples of both locations was decarbamoyl saxitoxin (dcSTX), but low concentrations of saxitoxin were also found in Manzanillo samples. Gonyautoxins GTX1, GTX2 and GTX3 were detected only at Guayacán, while in both locations, decarbamoylgonyatouxin (dcGTX2,3) toxins were detected. These findings represent the first time that causative toxins of PSP in Venezuela have been chemically identified, and confirm the presence of dcSTX and dcGTX in mussels from the Caribbean Sea. The presence of dcSTX and dcGTX in shellfish is indicative that Gymnodinium catenatum was a causative organism for outbreak of PSP.

Nucleotide profile of mouse liver: response to 2 ′- O-dibutyryl cytidene 3 ′,5 ′-cyclic monophosphate

The effect of 2 ′- O-dibutyryl cytidene 3 ′,5 ′-cyclic monophosphate (dibutyryl cCMP) on the nucleotide profile of mouse liver was examined. Dibutyryl cCMP caused an increase in the amount of CTP in mouse liver. Perchloric acid extracts of liver tissue were neutralized with tri- N-octylamine in trichlorotriflouroethene and, after removal of CLO 4 −, subjected to preliminary purification on a Cu 2+-loaded column of Chelex 100. A high-pressure liquid chromatographic anion-exchange procedure was used and gave good resolution of the free nucleotides on a single column.

Post-mortem redistribution of 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") in the rabbit. Part I: experimental approach after in vivo intravenous infusion.

Post-mortem redistribution is known to influence blood and tissue levels of various drugs. An animal model was used in an attempt to elucidate this problem for the amphetamine analogue, 3,4-methylenedioxymethamphetamine (MDMA). Rabbits received 1 mg/kg MDMA intravenously (iv) and were killed 2 h later in order to simulate the state of complete distribution in the body. MDMA and 3,4-methylenedioxyamphetamine (MDA) concentrations were determined in blood, urine, bile, vitreous humour, and various tissues (eye globe walls, brain, cardiac muscle, lungs, liver, kidneys, iliopsoas muscle and adipose tissue) using a high pressure liquid chromatographic (HPLC) procedure with fluorescence detection. In the first group (control group, sampling immediately post mortem) considerable MDMA concentrations were found in the brain and both lungs. In addition, our data indicate the elimination of MDMA by hepatic biotransformation and excretion via the bile. When the animals were preserved either 24 or 72 h post mortem (second group), an increase of MDMA and MDA levels in the liver and the eye globe walls was noticed. In the lungs, on the other hand, they tended to decline as a function of increasing post-mortem interval. MDMA levels in cardiac and iliopsoas muscle were fairly comparable and remained stable up to 72 h after death. In the third group, ligation of the large vessels around the heart took place immediately post mortem, but significant differences in blood and tissue MDMA concentrations between rabbits of group 2 and 3 could not be demonstrated. We therefore conclude that post-mortem redistribution of MDMA at the cellular level (viz. by pure diffusion gradient from higher to lower concentrations) is more important than its redistribution via the vascular pathway. Finally, MDA levels were relatively low in all samples, thus indicating that this is not a major metabolite in the rabbit, at least within the first 2 h after administration.

Uric acid is a genuine metabolite of Penicillium cyclopium and stimulates the expression of alkaloid biosynthesis in this fungus.

On searching for endogenous, low-molecular-weight effectors of benzodiazepine alkaloid biosynthesis in Penicillium cyclopium uric acid was isolated from ethanolic or autoclaved mycelial extracts of this fungus. The isolation was based on a three-step high-pressure liquid chromatography procedure guided by a microplate bioassay, and uric acid was identified by mass spectrometry and the uricase reaction. Conidiospore suspensions that were treated with this compound during the early phase of outgrowth developed emerged cultures with an enhanced rate of alkaloid production. Uric acid treatment did not increase the in vitro measurable activity of the rate-limiting biosynthetic enzyme, cyclopeptine synthetase. However, these cultures displayed a reduced rate of uptake of the alkaloid precursor L-phenylalanine into the vacuoles of the hyphal cells as assayed in situ. It is suggested that the depressed capacity of vacuolar uptake caused by the contact of outgrowing spores with uric acid liberated from hyphal cells results in an enhanced availability of the precursor L-phenylalanine in the cytoplasm and thus accounts at least in part for the increase in alkaloid production.

Distribution Study of 3,4-Methylenedioxymethamphetamine and 3,4-Methylenedioxyamphetamine in a Fatal Overdose

In this study, regional tissue distributions of the amphetamine analogue 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") and its metabolite 3,4-methylenedioxyamphetamine (MDA) in a fatal overdose are presented. Quantitation of MDMA and MDA levels occurred in blood samples taken centrally (right and left heart and main adjacent great vessels) and peripherally (subclavian and femoral blood). In addition, MDMA and MDA concentrations were determined in cardiac and iliopsoas muscle, both lungs, liver, both kidneys, spleen, the four brain lobes, cerebellum and brainstem, and adipose tissue. Finally, MDMA and MDA levels were determined in serum, vitreous humor, urine, and bile. For all samples, a fully validated high-pressure liquid chromatography procedure with fluorescence detection was used. The found substances were also identified with liquid chromatography-tandem mass spectrometry. Our data confirm that blood sampling from an isolated peripheral vein is recommended for MDMA and MDA. In addition, the vitreous humor MDMA level indicates that this fluid can be an interesting alternative when a suitable blood sample is missing. Considering the substantial differences in concentrations in blood samples taken from various sites in the body and the high levels in some tissues (e.g., in liver), we concluded that the influence of postmortem redistribution should be taken into account in the interpretation of toxicological data when an appropriate peripheral sample cannot be obtained or when blood samples are not available because of putrefaction.

A role for peroxisome proliferator-activated receptor alpha (PPARalpha ) in the control of cardiac malonyl-CoA levels: reduced fatty acid oxidation rates and increased glucose oxidation rates in the hearts of mice lacking PPARalpha are associated with higher concentrations of malonyl-CoA and reduced expression of malonyl-CoA decarboxylase.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear receptor transcription factor that has an important role in controlling cardiac metabolic gene expression. We determined whether mice lacking PPARalpha (PPARalpha (-/-) mice) have alterations in cardiac energy metabolism. Rates of palmitate oxidation were significantly decreased in isolated working hearts from PPARalpha (-/-) hearts compared with hearts from age-matched wild type mice (PPARalpha (+/+) mice), (62 +/- 12 versus 154 +/- 65 nmol/g dry weight/min, respectively, p < 0.05). This was compensated for by significant increases in the rates of glucose oxidation and glycolysis. The decreased fatty acid oxidation in PPARalpha (-/-) hearts was associated with increased levels of cardiac malonyl-CoA compared with PPARalpha (+/+) hearts (15.15 +/- 1.63 versus 7.37 +/- 1.31 nmol/g, dry weight, respectively, p < 0.05). Since malonyl-CoA is an important regulator of cardiac fatty acid oxidation, we also determined if the enzymes that control malonyl-CoA levels in the heart are under transcriptional control of PPARalpha. Expression of both mRNA and protein as well as the activity of malonyl-CoA decarboxylase, which degrades malonyl-CoA, were significantly decreased in the PPARalpha (-/-) hearts. In contrast, the expression and activity of acetyl-CoA carboxylase, which synthesizes malonyl-CoA and 5'-AMP-activated protein kinase, which regulates acetyl-CoA carboxylase, were not altered. Glucose transporter expression (GLUT1 and GLUT4) was not different between PPARalpha (-/-) and PPARalpha (+/+) hearts, suggesting that the increase in glycolysis and glucose oxidation in the PPARalpha null mice was not due to direct effects on glucose uptake but rather was occurring secondary to the decrease in fatty acid oxidation. This study demonstrates that PPARalpha is an important regulator of fatty acid oxidation in the heart and that this regulation of fatty acid oxidation may in part occur due to the transcriptional control of malonyl-CoA decarboxylase.

A rapid high-pressure liquid chromatographic procedure for determination of flumazenil in plasma

Introduction: Flumazenil antagonizes the effects of benzodiazepines at γ-aminobutyric acid (GABA) type A receptors in the central nervous system. Flumazenil has been reported to provoke panic attacks in patients with panic disorder (PD) but not in healthy controls. A rapid high-pressure liquid chromatographic (HPLC) method was developed for determination of flumazenil in plasma samples from PD patients receiving flumazenil and the results obtained with that assay are reported here. Methods: Samples from 37 PD subjects receiving 2 mg of flumazenil intravenously were analyzed. Extraction under basic conditions was followed by an HPLC assay with UV detection (250 nm). Lamotrigine was used as internal standard and a standard curve was constructed for each assay run. Flumazenil concentrations were measured in all the subjects in samples collected at 2 and 4 min after the drug administration and in some subjects, measurements were also done in samples collected at 7.5, 15, 30, 45, and 60 min. Results: The procedure was reproducible and linear (from 2.5 to 1000 ng/ml). At 2 and 4 min after flumazenil administration, the concentrations did not differ significantly between panicking and nonpanicking subjects, indicating that the pharmacokinetics of the drug is not the major determinant of the responses. There was a steep decline in the plasma concentration–time profile during the first 4 min, reflecting an extensive and rapid distribution after which the decline was slower. Discussion: The method described here is rapid, replicable, and convenient for the determination of flumazenil in plasma.

The Mass Transfer of 3, 5-Di-Tertiary-Butyl-4-Hydroxy Toluene and α-Tocopherol from Coextrusion Film Structures

The antioxidants, 3, 5-di-tertiary-butyl-4-hydroxy toluene (BHT) and α-tocopherol were incorporated into multilayer coextrusions consisting of a heat-seal layer, a core layer of high-density polyethylene (HDPE) impregnated with antioxidant (either BHT or α-tocopherol), and an outer HDPE layer. The rates of loss of the antioxidants from the respective films were determined as a function of time and temperature, using ultraviolet spectrophotometric and high-pressure liquid chromatographic procedures. Graphical analysis indicated that antioxidant loss from the coextrusion structures was well described by a first-order or pseudo-first-order rate expression. Volatilization from the respective surface layers was assumed to be the rate-limiting parameter for mass transfer. The results showed that for BHT, greater than 95% of the antioxidant was lost from the coextrusion within 36 h at 40°C, within 5 days at 30°C, and within 16 days at 23°C. In comparison, it is estimated that to achieve similar levels of loss of α-tocopherol from the coextrusion, it would require nearly 54 days at 30°C and 20 days at 40°C. At 23°C, 85% of α-tocopherol was lost from the coextrusion after 195 days of storage. Assuming a first-order rate expression, the rate-loss constants for BHT were 4.3 × 103, 29 × 103, and 65 × 103 h−1 at 23°C, 30°C, and 40°C, respectively. For α-tocopherol, the rate-loss constants were 0.4 × 103, 2.0 × 103, 6.8 × 103, and 28.4 × 103 h−1 at 23°C, 30°C, 40°C, and 50°C, respectively.

Cleaning level acceptance criteria and a high pressure liquid chromatography procedure for the assay of Meclizine Hydrochloride residue in swabs collected from pharmaceutical manufacturing equipment surfaces

A method using pharmacologically based and visual limit of detection criteria to determine the acceptable residue level for Meclizine Hydrochloride (MH) on pharmaceutical manufacturing equipment surfaces after cleaning is described. A formula was used in order to determine the pharmacologically safe cleaning level for MH. This level was termed as specific residual cleaning Level (SRCL) and calculated to be 50 μg 100 cm −2. The visual limit of detection (VLOD) was determined by spiking different levels of MH on stainless steel plates and having the plates examined by a group of observers. The lowest level that could be visually detected by the majority of the observers, 62.5 μg 100 cm −2, was considered as the VLOD for MH. The lower of the SRCL and VLOD values, i.e. 50 μg 100 cm −2, was therefore chosen as the cleaning acceptance criterion. A sensitive reversed-phase HPLC method was developed and validated for the assay of MH in swabs used to test equipment surfaces. Using this method, the mean recoveries of MH from spiked swabs and ‘180-Grit’ stainless steel plates were 87.0 and 89.5% with relative standard deviations (RSD) of ±3.3 and ±2.4%, respectively. The method was successfully applied to the assay of actual swab samples collected from the equipment surfaces. The stability of MH on stainless steel plates, on cleaning swabs and in the extraction solution was investigated.

Effect of Variety, Ultraviolet Light Exposure, and Enological Methods on thetrans-Resveratrol Level of Wine

Resveratrol is one of the wine components related to moderate wine consumption and reduction of serum cholesterol levels. The standard addition method was used with a high pressure liquid chromatography procedure to calculate resveratrol levels. Grape variety, UV light exposure, enzyme addition, skin contact time, and the fining agents, carbon and PVPP, affected the resveratrol levels of wines from the white varieties, Chardonnay and Chardonel, and the red varieties, Cabernet Sauvignon, Cynthiana, and Noble. UV light exposure of the grape clusters significantly increased the resveratrol level in Cynthiana and Noble wine produced in 1994, but did not yield a consistent result in the other experiments from the 1995 and 1996 wines. Enzyme addition significantly increased resveratrol levels in Chardonnay wine but not Chardonel wine. Skin contact time influenced the extraction of resveratrol from the skin in the red varieties, but the maximum extraction time was dependent on the variety of the grapes. Carbon addition decreased the resveratrol level in Noble wine but not in Cynthiana in the 1994 wines. The addition of PVPP significantly lowered resveratrol level in Chardonel, Chardonnay, Cabernet Sauvignon, Cynthiana, and Noble wine. However, low levels of added PVPP addition did not cause significant loss of resveratrol.

Effect of Regenerated Roots and Shoots on Nicotine Production in Tobacco Tissue Culture

The accumulation of nicotine in regenerated shoot and root tissues of Nicotiana tabacum L. cv MD 872 grown from callus culture has been examined by high pressure liquid chromatography (HPLC) procedures. The presence of roots on callus tissue dramatically increased the accumulation of nicotine in both callus without shoots and in callus which had regenerated shoots. When shoots were regenerated, the concentration of nicotine that accumulated in callus decreased whether roots were present or not. When varying concentrations of the nicotine precursors ornithine and putrescine were fed to callus derived tissue with and without regenerated roots, the rooted tissue was able to take up the precursors and accumulate nicotine in the shoots in amounts greater than controls. The half-life turnover rate for ’H-nicotine absorbed by the roots and transported into shoots was approximately 48 hours. These experiments indicate that although the root is the major site of synthesis of nicotine in tobacco, the accumulation of nicotine in various parts of the plant is regulated by the interaction of both root and shoot tissue.

A simple gel electrophoretic method for analyzing the muropeptide composition of bacterial peptidoglycan.

The muropeptide composition of bacterial peptidoglycan is currently most efficiently determined by reverse-phase high-pressure liquid chromatography (HPLC). Though sensitive, the HPLC procedure is technically demanding and has been applied to a relatively small number of bacterial strains and species. We have found that fluorescence-assisted carbohydrate electrophoresis (FACE) is a simple, rapid method by which reducing muropeptides from multiple peptidoglycan samples can be visualized. Individual reducing muropeptides were covalently labeled with the fluorescent molecule 8-aminonaphthalene-1,3,6-trisulfonic acid, after which they were separated by electrophoresis through a 35% polyacrylamide gel and visualized by exposure to UV light. FACE detected the appropriate numbers of reducing muropeptides in the proper proportions for four bacteria: Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, and Yersinia enterocolitica. As little as 2 to 5 pmol per muropeptide was detected when the intensity of the fluorescent signal was measured with a charge-coupled device camera, at a level of sensitivity between 50 and 250 times higher than that of the classic HPLC technique. Thus, FACE may be used to identify interesting peptidoglycan samples prior to more-extensive analysis by HPLC, or FACE may eventually replace HPLC for some applications.