Pre Column Research Articles

ثابت سرعة تحلل بعض مشتقات الامينوكلايكوسيدات

Amino glycoside derivation including, Neomycin, Streptomycin, Kanamycin and Gentamycin with special reagents, which are benzoylchloride; benzene sulfonyl chloride and phthalic anhydride were made to enhance Uv-detectability for HPLC analysis. But there are many problems facing pre column derivation and in order to solve this, the conductivity of antibiotic derivatives were used to calculate the dissociation constant and the hydrolysis rate which determined concern type reaction. In addition the characteristics those controlling the hydrolysis of antibiotic-derivatives were investigated.

Variation in Amino Acids Composition through Pre column Derivatisation using Phenylisothiocyanate by HPLC in Some Economically Important Less Explored Wild Allium Species of Western Himalayas

Less explored Allium species are being used as green vegetables and as a condiment by the local inhabitant of high-altitude areas of Uttarakhand. In the present study, four economically important less explored wild Allium species viz. A. auriculatum, A. ampeloprasum, A. ascalonicum and A. rubellum have screened for their amino acid contents by High-performance liquid chromatography. The hydrochloric acid hydrolysate of these four species, the Allium amino acids were derivatised with phenylisothiocyanate resulting phenylthiocarbamyl derivatives and separated on a reverse-phase column by gradient elution with aqueous buffer and acetonitrile-water (60:40 v/v) and detected in UV region at 254 nm. The Pico-tag (3.9 × 300 mm) C18 column equilibrated with the solvents. The elution of all amino acid derivatives was achieved in 12 min using gradient elution by increasing concentration of aqueous buffer and acetonitrile-water. Total seventeen amino acids were present in these Allium species. The ratio of essential amino acids to total amino acids found 1:2.14 in Allium auriculatum, 1:2.35 in Allium ampeloprasum, 1:1.38 in A. ascalonicum and 1:3.44 in Allium rubellum. These less explored Allium species contained substantial amount of essential and non-essential amino acids. Among these Allium species, Allium auriculatum and Allium rubellum found most promising as far as essential and non-essential amino acids composition concerned.

High performance liquid chromatographic separation of platinum (II), gold (III), vanadium (IV), vanadium (V), molybdenum (VI) and analysis of cis-platin as platinum (II) in cis-plasol injection, urine, and blood serum using pyridoxal-4-phenyl-3-thiosemicarbazone as complexing reagent

A high performance liquid chromatographic (HPLC) method has been proposed for separation of Pt (II), Au (III), V (IV)/V (V), Mo (VI) and analysis of cis-platin on the basis of pre column derivatization using pyridoxal-4-phenyl-3-thiosemicarbazone (PDPTSC) as complexing reagent. The complexes were extracted in trichloromethane or n-butanol and were separated from Kromacil100, C-18, 10 µm (250 cm × 4.6 mm, i.d) column with a ternary mixture of acetonitrile:water:methanol (20:30:50 V/V/V) with a flow rate of 1 mL.min−1 and ultra-violet detection was at 320 nm. The linear calibration was achieved with 0.5–5 µg.mL−1 and detection limit of 10 ng.mL−1 platinum (II). The determination of cis-platin (cis-plasol) injection by HPLC method showed RSD 2.41%. The developed method was employed for the analysis of cis-platin from urine and blood serum of cancer patients after chemotherapy and platinum contents were found 47.0–87.0 ng.mL−1 and 77.4–424 ng.mL−1 with RSD within 0.824–3.42% for urine and 0.421–3.84% for serum. The recovery of platinum (II) was found as 98.1% with RSD 1.99%.

Assay of Desmopressin Acetate in Nasal Spray: Development of Validated Pre Column HPLC-Fluorescence Method.

Purpose: Desmopressin acetate (DDAPV), a synthetic analogue of vasopressin, has been recommended to be used in diabetes insipidus, mild forms of hemophilia and Von Willebrand disease. The DDAPV is available for adminstration via different routes viz. oral, parenteral and nasal, however its dose is very less in case of nasal sprays (20 µg) and parenteral route (4 µg) compared to oral route (0.1 to 0.3 mg in tablet). A sensitive and selective method is needed to be developed and validated for assay of low concentrations of DDAPV in its pharmaceutical dosage form i.e. nasal spray.Methods: Simple and specific HPLC-Fluorescecne method has been proposed for the quantitation of DDAPV at nanogram level in nasal formulations for the first time. DAPV, DDAPV EP impurity-B, chlorobutanol, benzalkonoium chloride were successfully derivatised with Ortho-Phthalaldehyde (OPA) and co-eluted on a C8 (50×2.1 mm, 3.5 µm particle size, 120Å) with mobile phase composed of 0.1% trifluroacetic acid, acetonitrile and Isopropyl alcohol in ratio of 70:25:5. The emission was measured at 450nm and flow rate was 0.8ml/min. The reaction was optimized in the terms of pH, stability of formed fluorophore and time consumed during the reaction.Results: The maximal fluorescence intensity was reached when the solutions were mixed for 3 min, and remained constant for at least 30 min at 20-25ºC. The calibration curve was found linear from 50 to 5000 ng/ml with weight of 1/X2. The limit of detection was 10ng/ml and precision was less than 2.0.Conclusion: The developed HPLC-fluorescence assay method was successfully applied for quantitation of DDAPV in nasal spray. HPLC-Fluorescence method was specific, sensitive, precise and accurate for determination of DDAPV. The method was able to quantify DDAPV at 50ng/ml with sufficient accuracy and precision. The validated HPLC-Fluorescence was successfully applied.

Preparation of Magnetic Microsphere‐Gold Nanoparticle‐Immobilized Enzyme Batch Reactor and Its Application to Enzyme Inhibitor Screening in Natural Extracts by Capillary Electrophoresis

A novel strategy for screening the enzyme inhibitors from the natural products by capillary electrophoresis (CE) with a pre column enzyme batch reactor prepared by magnetic microsphere (MB)‐gold nanoparticles (AuNPs) is reported. The enzyme batch reactor was prepared by immobilizing the enzymes on the MB conjugated AuNPs (MB‐AuNPs). To demonstrate this strategy, xanthine oxidase (XOD) was employed as a model for the activity of the enzyme, inhibition study, and inhibitor screening. With the developed CE method, the enzyme activity was determined by the quantification of the peak area of the product. Enzyme inhibition can be read out directly from the reduced peak area of product in comparison to a reference electropherogram obtained in the absence of any inhibitor. A statistical parameter Z‘ factor was recommended for evaluation of the accuracy of a drug screening system. In the present study, it was calculated to be 0.7, implying a good accuracy. The screening of two natural extracts from Cortex Phellodendri and Rhizoma Galangae showed that they were positive for XOD inhibition by the present method. Using this immobilized enzyme technology combined with CE separation not only provides the advantages such as convenience, rapidity and low cost, but also provides a new platform for discovering enzyme‐inhibitor drug lead compounds.

HPLC Determination of Metformin, Famotidine and Ranitidine by Derivatization with Benzoin from Drugs and Biological Samples

A novel High Performance Liquid Chromatography (HPLC) method has been developed based on pre column derivatization with benzoin for determination of metformin, famotidine and ranitidine. The separation was achieved from C18 column when eluted isocratically, the solution of the drugs with methanol, water, acetonitrile and Tetra Hydrofuran (THF) (40:40:16:4 v/v) with a flow rate at 1 mL/min. UV detection was found to be 268 nm. Linear calibrations range was obtained with 2.5-12.5 μg/ml, with limits of detection (LODs) 0.091-0.30 μg/ml. The total run time for elution was 3.5 min. The derivitization, separation and quantitation was repeatable (n=4) in terms of retention time and peak height/peak area with Relative Standard Deviation (RSD) within 0.84-1.55% and 0.68-1.17% respectively. The method was applied for the analysis of metformin, famotidine and ranitidine from pharmaceutical preparations, human serum and human urine. The possible interfering effects of the sample matrix were checked by the analysis by standard addition method and matrix effect was not indicated.

A Non-Invasive Glucose Estimation in Saliva Samples by using Pre Column Derivatised RP-HPLC

Diabetes is characterized by polyurea, polydypsia and weight loss in spite of polyphagia, hyperglycemia, glucosuria, ketosis, and acidosis and coma. The cause of clinical diabetes is always due to deficiency of the effects of insulin at the tissue level, but the deficiency may be relative. One of the common forms Type-1 (Insulin Dependent Diabetes Mellitus IDDM) is due to insulin deficiency caused by auto-immune destruction of β-cells in pancreatic islets, the A, D and F cells remain intact. Type 1 diabetes usually develops before the age of 40 and hence called juvenile diabetes [1-3].

B-4. 9-アンスリルジアゾメタンを利用したプロスタグランディンの高速液体クロマトグラフィー

B-4. 9-アンスリルジアゾメタンを利用したプロスタグランディンの高速液体クロマトグラフィー

Effect Of A Dialysis Session On Plasma Branched Chain Aminio Acids In Hemodialysis Patients

AbstractProtein and amino acid (AA) metabolism is abnormal in End stage renal disease (ESRD). Hemodialysis (HD) procedure is a strong catabolic stimulus. Branched chain amino acids (BCAAs) can affect other AA levels by reducing AA efflux from muscle due to inhibition of muscle protein degradation. Essential amino acids and keto acid supplements including BCAA and branched-chain keto acid (BCKA) are proposed to decrease protein intake while maintaining protein status. This study was taken up to evaluate the effect of a dialysis session on plasma BCAA’s for which fifteen patients of ESRD on Maintenance HD, thrice a week were recruited into the study. Analysis was done on samples drawn at the beginning (pre-HD) and after the end of each dialysis session (post-HD). Plasma BCAA’s were estimated by Reverse phase High performance liquid chromatography using pre column derivatization with O-pthalaldehyde-Mercaptoethanol. A significant decrease in plasma concentration of Valine and Isoleucine were observed post-HD compared to the pre-HD levels (p<0.05). After correcting the data by creatinine, the decrease in plasma concentrations of Valine and Isoleucine were still found to be statistically significant. The percentage losses after the completion of HD were –24.45, –23.19, and –6.22% respectively for valine, isoleucine, and leucine. The lower reduction in leucine could be due to its appearance from muscle catabolism during the dialysis session. In conclusion, hemodialysis itself may influence dialysate amino acid losses and may have an effect on muscle protein breakdown and this negative protein can be reversed with nutritional supplementation.

Validated stability indicating liquid chromatographic determination of ebastine in pharmaceuticals after pre column derivatization: Application to tablets and content uniformity testing

An accurate, simple, sensitive and selective reversed phase liquid chromatographic method has been developed for the determination of ebastine in its pharmaceutical preparations. The proposed method depends on the complexation ability of the studied drug with Zn2+ ions. Reversed phase chromatography was conducted using an ODS C18 (150 × 4.6 mm id) stainless steel column at ambient temperature with UV-detection at 260 nm. A mobile phase containing 0.025%w/v Zn2+ in a mixture of (acetonitril/methanol; 1/4) and Britton Robinson buffer (65:35, v/v) adjusted to pH 4.2, has been used for the determination of ebastine at a flow rate of 1 ml/min. The calibration curve was rectilinear over the concentration range of 0.3 - 6.0 μg/ml with a detection limit (LOD) of 0.13 μg/ml, and quantification limit (LOQ) of 0.26 μg/ml. The proposed method was successfully applied for the analysis of ebastine in its dosage forms, the obtained results were favorably compared with those obtained by a comparison method. Furthermore, content uniformity testing of the studied pharmaceutical formulations was also conducted. The composition of the complex as well as its stability constant was also investigated. Moreover, the proposed method was found to be a stability indicating one and was utilized to investigate the kinetics of alkaline and ultraviolet induced degradation of the drug. The first-order rate constant and half life of the degradation products were calculated.

Asymmetric dimethylarginine, cortisol/cortisone ratio, and C-peptide: Markers for diabetes and cardiovascular risk?

Diabetes and prediabetic conditions are growing cardiovascular risk factors. Better understanding and earlier recognition and treatment of dysglycemia-related risk are health priorities. We assessed the predictive value of 3 proposed new markers for diabetes and cardiovascular risk. We tested whether the plasma levels of (1) asymmetric dimethylarginine (ADMA), (2) cortisol/cortisone (Cl/Cn) ratio, and (3) C-peptide predicted glycemic status, coronary artery disease, and death or myocardial infarction (MI) in a nested case-control cohort (N = 850) with normal fasting glucose (< 110 mg/dL), impaired fasting glucose (110-125), or diabetic (> or = 126) status. High-sensitivity C-reactive protein (hsCRP) served as a control risk marker. Follow-up averaged 2.6 +/- 1.4 years. High-pressure liquid chromatography with pre-column derivitization and fluorescence was used to assay ADMA, liquid chromatography/tandem mass spectrometry for Cl and Cn, and chemiluminescent immunoassay for C-peptide. Asymmetric dimethylarginine levels were positively associated with glycemic category (P < .001). Quartiles 2 to 4 ADMA also conferred increased risk of death/MI independent of hsCRP and other risk factors (adjusted hazard ratio, 2.1; P = .002). Cortisol/Cortisone ratios (P = .013) and C-peptide (P = .047) were associated with glycemic categories but less strongly than ADMA. Quartiles 2 to 4 Cl/Cn were protective against incident death/MI (adjusted hazard ratio, 0.48; P < .001), whereas C-peptide did not predict outcomes. Among a high coronary risk case-control cohort, ADMA (strongly), Cl/Cn (moderately), and C-peptide (weakly) predicted glycemic categories. Asymmetric dimethylarginine and Cl/Cn also predicted clinical outcome independent of and more strongly than hsCRP. Asymmetric dimethylarginine and Cl/Cn represent promising new candidate markers of dysglycemia and associated cardiovascular risk.

Simultaneous determination of levofloxacin, gatifloxacin and moxifloxacin in serum by liquid chromatography with column switching

Liquid chromatography with a column-switching technique was developed for simultaneous direct quantification of levofloxacin, gatifloxacin and moxifloxacin in human serum. Serum samples were injected on a LiChroCART ® 4-4 pre-column (PC) filled with a LiChrospher ® 100 RP-18, 5 μm where fluoroquinolones (FQs) were purified and concentrated. The FQs were back-flushed from the PC and then separated on a Supelcosil ABZ+ Plus (150 mm × 4.6 mm i.d.) analytical column with a mobile phase containing 10 mM phosphate buffer (pH 2.5), acetonitrile (88:12, v/v) and 2 mM tetrabutyl ammonium bromide. The effects of ion-pair reagents, buffer type, pH and acetonitrile concentrations in the mobile phase on the separation of the three FQs were investigated. Fluorescence detection provided sufficient sensitivity to achieve a quantification limit of 125 ng/ml for levofloxacin and moxifloxacin; 162.5 ng/ml for gatifloxacin with a 5 μl sample size. The on-line process of extraction avoids time-consuming treatment of the samples before injection and run time is shortened. The recovery, selectivity, linearity, precision and accuracy of the method are convenient for pharmacokinetic studies or routine assays.

Simple determination of terbutaline in dog plasma by column-switching liquid chromatography

Terbutaline is a β-adrenergic receptor antagonist that acts as a bronchodilator in the treatment of asthma and chronic bronchitis. In the present work, a column-switching high-performance liquid chromatographic method was developed to monitor terbutaline sulphate in dog plasma. The system consists of a C 2 pre-column (PC) and a C 18 analytical column connected in series via a switching valve. Atenolol was used as the internal standard. Good linearity was achieved in the range of 5–800 ng/ml plasma. The mean intra- and inter-assay variation coefficients for this analysis were 2.3 and 4.7%, respectively. The average recovery for terbutaline was 87.4% from plasma. The mean concentration after three freeze-thaw cycles was 99.4% of the normal value. The analytical sensitivity and accuracy of this assay is adequate for characterisation of the pharmacokinetics of oral administration of terbutaline to dogs and has been successfully used to provide pharmacokinetic data using pulsatile and immediate-release tablets.

Detection of pharmaceutical contaminations of river, pond, and tap water from Cologne (Germany) and surroundings

A method for the simultaneous determination of polar pharmaceutical compounds in water is presented. Samples from 27 rivers and ponds were investigated for contents of Gemfibrocil, a lipid blood regulating compound, Clofibric acid, a metabolite of lipid regulating compounds (Clofibrat, Etofibrat, Etofyllinclofibrat), antirheumatics (Diclofenac, Ibuprofen, Ketoprofen, Indomethacin, Fenoprofen) and Sarkosin-N-(phenylsulfonyl) (SPS), a metabolite of a corrosion inhibiting agent. In addition water samples were investigated for some main metabolites of Ibuprofen, 2-[4-(2-hydroxy-2-methylpropyl)phenyl]propionic acid (hydroxy-ibuprofen) and 2-[4-(2-carboxypropyl)phenyl]propionic acid (carboxy-ibuprofen), which are excreted after oral intake. For gas chromatographic analysis the drugs were converted into their methyl-derivatives by "on column" reaction with TMSH, TMAH and "pre column" with diazomethane. The detection limits with TMSH were in the same range or lower than those with diazomethane or TMAH. The compound most frequently found was SPS which was only absent in waters with natural surroundings. Diclofenac could be detected in 10 out of 27 water samples in concentrations of up to 15 micrograms/l. In contrast, none of the pharmaceuticals investigated were present in 8 drinking water samples from Cologne and surroundings. Though concentrations measured were far below pharmacological doses, the data suggest a steady flux of these drugs into the environment.

Study on volatile components in salami by reverse carrier gas headspace gas chromatography–mass spectrometry

Salami are a typical seasoned sausage of Italy; a number of types are produced, according to local traditional recipes. As industrial production has taken place, a number of problems rise in obtaining products similar to the traditional ones. The use of selected microbial starters is permitted by Italian law for some years and at present, micro biological research is engaged in selecting starters similar to the ones isolated from traditional products, with the aim of obtaining organoleptic characteristics close to the ones of traditional recipes. A study was carried out concerning the characterisation of volatile components of salami by headspace capillary gas chromatography–mass spectrometry. As during the sampling step, analytes could reach the analytical column, the carrier gas rate was back flushed in the latter, while a pre column was used as cold trap. Then GC–MS analysis follows. By these techniques, we were able to highlight typical profiles of different salami, as well as monitoring the ripening of a traditional and a starter added salami. Main peaks are of fermentative origin, while also peaks from spices were detected. Ethyl propionate was used as internal standard to be able to normalise the peaks amounts.

Determination of 13- cis-3-hydroxyretinoic acid, all- trans-3-hydroxyretinoic acid and their 4-oxo metabolites in human and animal plasma by high-performance liquid chromatography with automated column switching and UV detection

A high-performance liquid chromatographic method with automated column switching was developed for the simultaneous determination of 13- cis-3-hydroxyretinoic acid, all- trans-3-hydroxyretinoic acid and their metabolites 13- cis-3-hydroxy-4-oxo-retinoic acid and all- trans-3-hydroxy-4-oxo-retinoic acid in plasma samples from man, rat, dog, rabbit and mouse. The method consists of deproteination of plasma (0.4 ml) with ethanol (1.5 ml), containing the internal standard Ro 12-7310. After centrifugation, 1.4 ml of the supernatant was directly injected onto the precolumn (PC) (4×4 mm) packed with LiChrospher 100 RP-18 (5 μm). Ammonium acetate (0.02%)-acetic acid-ethanol (100:3:4, v/v/v) was used as mobile phase M1A during injection, as well as to decrease the elution strength of the injection solution by on-line addition using a T-piece (M1B). After valve switching, the retained components were transferred to the analytical column (AC), separated by gradient elution and detected at 360 nm. Two coupled Purospher 100 RP-18 endcapped columns (both 250×4 mm) were used for the separation, together with a mobile phase consisting of acetonitrile-water-10% ammonium acetate-acetic acid, (A), 540:450:2:30 (v/v/v/v), (B), 600:350:2:30 (v/v/v/v), and (C), 950:40:2:30 (v/v/v/v). The method was linear in the range 1–500 ng ml −1, at least, with a quantification limit of 1 ng ml −1. The mean recoveries from human plasma were 100–107% and the mean inter-assay precision was 2.0–4.7% (range 1–500 ng ml −1). Similar results were obtained for animal plasma. The analytes were stable in the plasma of all investigated species stored at −20°C for 3 months, at least. The method was successfully applied to clinical and toxicokinetic studies.

Anaerobic uptake of glutamate and aspartate by enhanced biological phosphorus removal activated sludge

Mechanisms of the anaerobic uptake of glutamate and aspartate by enhanced biological phosphorus removal activated sludge were investigated. Sludge hydrolysate with hydrochloric acid was analyzed by reverse phase HPLC after pre column derivatization with dabsyl-chloride. The experimental results indicated that glutamate is accumulated as a polymer consisting of γ-aminobutyric acid and an unknown amino acid. On the other hand, aspartate was found to be deaminated and then accumulated as PHA.

Prevention of co-elution of steroid sulfates with serum proteins from pre-column in column-switching HPLC system.

A method to prevent co-elution of steroid sulfates with proteins in serum from the pre-column in column-switching HPLC was developed. The pre-column, a polymer-coated mixed function column, was used for ion-pair chromatography with 5 mM tetra-n-butylammonium (TBA) ion. As steroid sulfates, estriol 3-sulfate, dehydroepiandrosterone 3-sulfate and pregnenolone 3-sulfate were used. Human serum (25 microl) was diluted with mobile phases including 5, 100 and 500 mM TBA ion, and then injected directly into the pre-column. The peak areas of the steroid sulfates in serum samples were compared with those of the steroid standards without serum. When 25/microl of serum was diluted with mobile phase including 100 or 500 mM TBA ion, the steroid sulfates in serum were retained in the pre-column; however, the steroid sulfates from the same sample diluted with mobile phase containing 5 mM TBA ion were not retained in the pre-column. Addition of an excess amount of counter ion (TBA ion) into the serum sample made it possible to retain the steroid sulfates in the pre-column. This method was applied to column-switching HPLC for measurement of steroid sulfates in serum using a semi-microcolumn as the analytical column.

Anaerobic uptake of glutamate and aspartate by enhanced biological phosphorus removal activated sludge

Mechanisms of the anaerobic uptake of glutamate and aspartate by enhanced biological phosphorus removal activated sludge were investigated. Sludge hydrolysate with hydrochloric acid was analyzed by reverse phase HPLC after pre column derivatization with dabsyl-chloride. The experimental results indicated that glutamate is accumulated as a polymer consisting of γ-aminobutyric acid and an unknown amino acid. On the other hand, aspartate was found to be deaminated and then accumulated as PHA.

On‐line derivatization of peptides for improved sequence analysis by micro‐column liquid chromatography coupled with electrospray ionization‐tandem mass spectrometry

An on-line method has been developed for the derivatization and coupled liquid chromatogrpahy (LC)/electrospray ionization (ESI) MS analysis of peptides at the femtomol level. Peptides are reacted with N-succinimidyl-2(3-pyridyl)acetate (SPA) in buffered aqueous medium at pH 7 following loading on a precolumn (PC) in a microcolumn switching system. The fast-hydrolysing reagent is dissolved in dry methanol and mixed, in a 3 vol% ratio, with a buffer just before reaching the sample on the PC. Following the reaction and wash, the N-pyridylacetyl (PA) derivatives formed are transferred to the analytical micro-high-performance (HP) LC column for separation and subsequent ESI tandem MS analysis. The reaction is nearly quantitative and takes place selectively at the N-terminal and lysine amino functions, the latter providing a chemical means to distinguish between isobaric residues lysine and glutamine. In some cases, a minor reaction was observed with the tyrosine hydroxyl group. The N-terminal PA group was able to alter the collision-induced fragmentation pathway of peptides in favour of the formation of abundant b-type ions, a helpful feature for sequence elucidation of unknown peptides, particularly with quadrupole ion trap instruments. © 1997 John Wiley & Sons, Ltd.