High-pressure Liquid Chromatographic Method Research Articles

Quantitative analysis of lycorine in Galanthus alpinus Sosn. var. alpinus by HPLC-DAD

In this study, a reversed-phase, high pressure liquid chromatography (RP-HPLC) method was applied in the quantitative analysis of lycorine in the aerial parts and bulbs of Galanthus alpinus Sosn. var. alpinus collected from Rize during the flowering and fruiting seasons. A simple method to extract lycorine from the plant specimens was performed, using pre-packed columns with diatomaceous earth (Extrelut®). The chromatographic separation was achieved employing an isocratic system with a mobile phase including trifluoroacetic acid-water-acetonitrile (0.01:95:5) measured at a flow rate of 1 mL/min using a diode array detector (DAD). The lycorine content in the bulbs during the flowering and fruiting seasons was detected as 0.01576 and 0.02351 %, respectively. Also, validation studies showed that the method was specific, accurate and precise.

Isolation, Identification and Characterization of Degradation Impurity of Atorvastatin in Fixed Dose Combination of Atorvastatin and Ezetimibe

The objective of this study is to isolation and characterization of unknown degradation product of Atorvastatin calcium in combination formulation product with Ezetimibe by using modern techniques of separation and aracterization. An unknown impurity is generating during a forced degradation study of Atorvastatin and Ezetimibe fixed-dose combination tablets. By using the gradient reversed-phase high-pressure liquid chromatographic method, unknown degradation impurity was detected and quantified in the range of 0.05% to 0.2% of Atorvastatin. The impurity was enriched by extreme oxidation degradation of Atorvastatin and isolated through preparative HPLC. The structure of the impurity was characterized by mass and NMR spectrum.

Acacia catechu (L.f.) Willd and Scutellaria baicalensis Georgi extracts suppress LPS-induced pro-inflammatory responses through NF-кB, MAPK, and PI3K-Akt signaling pathways in alveolar epithelial type II cells.

Acacia catechu (L.f.) Willd (ACW) and Scutellaria baicalensis Georgi (SBG) are one of the most famous couplet Chinese medicines, widely used for treating infantile cough, phlegm, and fever caused by pulmonary infection. However, the underlying molecular mechanism of their anti-inflammatory activity has not been determined. The aim of this study was to evaluate the protective effect of this couplet Chinese medicines (ACW-SBG) on lipopolysaccharide (LPS)-induced inflammatory responses in acute lung injury (ALI) model of rats and the potential molecular mechanisms responsible for anti-inflammatory activities in alveolar epithelial type II cells (AEC-II). Standardization of the 70% ethanol extract of ACW and SBG was performed by using a validated reversed-phase high-pressure liquid chromatography method. Rats were pretreated with ACW-SBG for 7days prior to LPS challenge. We assessed the effects of ACW-SBG on the LPS-induced production of tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) in the bronchoalveolar lavage fluid (BALF). The wet-to-dry weight ratio was calculated, and hematoxylin and eosin staining of lung tissue was performed. Cell viability of AEC-II was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Real-time quantitative reverse transcription polymerase chain reaction assay was carried out to quantify the relative gene expression of TNF-α and IL-1β in AEC-II. The western blotting analysis was executed to elucidate the expression of mediators linked to nuclear factor-kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and phosphatidylinositol-3 kinase-protein kinase B (PI3K-Akt) signaling pathways. ACW-SBG significantly decreased lung wet-to-dry weight ratio, ameliorated LPS-induced lung histopathological changes, and reduced the release of inflammatory mediators such as TNF-α and IL-1β in BALF. In AEC-II, we found that the expression of TNF-α mRNA was also inhibited by ACW-SBG. ACW-SBG blocked NF-κB activation by preventing the phosphorylation of NF-κB (p65) as well as the phosphorylation and degradation of the inhibitor of kappa B kinase. ACW-SBG extracts also inhibited the phosphorylation of respective MAPKs (c-Jun N-terminal kinase, extracellular signal-regulated kinase, and p38) as well as Akt. The present study demonstrated that ACW-SBG played a potent anti-inflammatory role in LPS-induced ALI in rats. The potential molecular mechanism was involved in attenuating the NF-κB, MAPKs, and PI3K-Akt signaling pathways in LPS-induced AEC-II.

Effect of bromhexine on the pharmacokinetic of tilmicosin in broiler chickens

Concurrent administration of drugs may alter their pharmacokinetic parameters, so; investigation to what extent bromhexine hydrochloride affects the pharmacokinetic behavior of tilmicosin was our aim of this work. Ten broiler chickens were classified into two groups as follow, the first one (tilmicosin group) was given single oral dose of tilmicosin (20 mg/kg.b.wt.) while the 2nd (pre-treated group) was given single oral dose of bromhexine hydrochloride (1 mg/kg.b.wt.) followed by single oral dose of tilmicosin (20 mg/kg.b.wt.) one hour later. The serum concentration of tilmicosin was measured using High Pressure Liquid Chromatography (HPLC) method. The results revealed that the mean serum concentrations of tilmicosin were significantly lower in pre-treated group when compared with tilmicosin alone group at the corresponding time intervals. Pharmacokinetic parameters were significantly differed (p<0.001) between both groups. The maximum serum concentration were (Cmax0.70±0.02, 0.81±0.04µg/ml), achieved at Tmax of (tmax 0.89±0.16, and 2.10±0.06h), absorption half-life (t0.5ab) of 0.16±0.08, and 0.37±0.01 hour, area under curve (AUC) of 12.96±0.42 and 16.73±0.42µg.h/ml) in tilmicosin-bromhexine and tilmicosin alone groups respectively. In conclusion, based on the obtained pharmacokinetic parameters, these findings showed that bromhexine accelerates the tilmicosin penetration into body tissues, achieving higher and faster concentrations than when given tilmicosin alone.

Oxidative DNA Damage and NOX4 Levels in Children with Undescended Testes.

Undescended testis (UDT) is a common urological disorder. Patients with UDT have a risk of malignancy and infertility. The development of these conditions may be due to oxidative stress mediated by reactive oxygen species. The aim of this study was to investigate the relationship between these parameters by detecting oxidative DNA damage (8-hydroxy 2 deoxyguanosine/106 deoxyguanosine), ischemia-modified albumin (IMA), malondialdehyde (MDA), and nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) levels in children with UDT and healthy control group. The blood samples were obtained from 30 patients with UDT and 40 healthy male subjects. The levels of oxidative DNA damage were detected by high-pressure liquid chromatography method. We used commercially available kits that use enzyme-linked immunosorbent assay method to measure IMA, MDA, and NOX4 levels. The levels of MDA, IMA, NOX4, and oxidative DNA damage in children with UDT were statistically significantly higher than control group. In addition, we found that the levels of NOX4, IMA, and oxidative DNA damage after 12 months of age was significantly higher than before 12 months of age. We identified increased lipid peroxidation, oxidative DNA damage, IMA, and NOX4 levels in children with UDT. Delay in the treatment of UDT may cause oxidative damage. That is why, according to us the antioxidant treatment may be beneficial in children with UDT.

Coupling mesoporous imprinted polymer based DGT passive samplers and HPLC: A new tool for in-situ selective measurement of low concentration tetrabromobisphenol A in freshwaters

Accurate measurement of tetrabromobisphenol A (TBBPA) is very important because of its widespread environmental pollution. Diffusive gradients in thin films technique (DGT), an in-situ passive sampling method, is regarded as a reliable and robust measurement technique. A new DGT technique based on mesoporous imprinted polymer was combined with high-pressure liquid chromatography (HPLC) method for sampling, preconcentration and monitoring low concentration TBBPA in natural waters. The diffusion coefficient of TBBPA through the diffusive gel was measured by diffusion cell test and simulated using mathematical expression. The effects of different ambient conditions were tested under laboratory conditions and the performance of DGT sampler was validated in natural waters. The diffusion coefficient of TBBPA in the diffusive gel was measured as 2.18 × 10−6 cm2 s−1 and simulated as in the range 1.41–3.48 × 10−6 cm2 s−1 by Amsden model. Comparison of experimental and theoretical data, the validity of the experimental method can be verified by the mathematical model. The binding agent with mesoporous imprinted polymer showed selective affinity to TBBPA and its adsorption rate met the requirement of DGT device. The DGT method detection limit was at the level of ng L−1 for 7 days deployment. DGT sampler was suitable for application in aquatic environment with a range of pH (4.5–7.6), ionic strength (1 × 10−4 - 0.5 mol L−1), and dissolved organic matter (DOM) concentration (0–10 mg C L−1). The coupling method of DGT and HPLC was a promising technique for in situ sampling, preconcentration and monitoring low concentration TBBPA in most typical natural waters.

Sabella spallanzanii mucus contain a galactose-binding lectin able to agglutinate bacteria. Purification and characterization

Lectins are present in almost all living organisms and are involved in several biological processes, including immune responses. In the present study, a calcium dependent galactose-binding lectin exhibiting an apparent MW of 43 kDa has been characterized and purified from the mucus of the polychaete Sabella spallanzanii by using both affinity chromatography and high-pressure liquid chromatographic methods. Its agglutinating activity towards rabbit erythrocytes was significantly modified by the addition of calcium or EDTA. The activity was optimal at temperature values comprised between 4 and 18 °C, maintain a 50% of activity between 20 and 37 °C, was significant deleted after exposure at 50 °C, and was depleted at 90 °C. The S. spallanzanii Galactose-Binding Lectin (SsGBL) was able to agglutinate bacteria and to preferentially recognize Gram-negative bacteria. The strongest agglutinating activity was observed towards Vibrio alginolyticus and Escherichia coli, by contrast mucus agglutinated in a lesser extent both Aeromonas hydrophyla and the Gram-positive Micrococcus lysodeikticus thus suggesting its involvement in host pathogen interactions.

Extraction and quantification of phycobiliproteins from the red alga Furcellaria lumbricalis

Phycobiliproteins are natural additives in food industry and essential fluorescent probes in biotechnology. Various algal species may be suitable for their extraction, therefore, it is important to study different seaweeds to determine the highest-yielding species. Furcellaria lumbricalis, a red algal species primarily harvested from Canadian, Danish and Estonian waters, has been utilized for merely furcellaran extraction in Estonia on a commercial scale for over 50 years. To further valorize the bioresource it is vital to study whether additional products, such as phycobiliproteins, can be extracted. In the given study, optimal phycobiliprotein extraction conditions from F. lumbricalis were determined for the first time. For quantification, a novel high-pressure liquid chromatography method with fluorescence and photodiode array detection was developed. Considering R-phycoerythrin, citrate pH 6 buffer gave the highest yields (0.13% using fluorescence detector and 0.43% using photodiode array detector) after an extraction for 24 h at 20 °C. Different enzymes (cellulases, xylanases, galactosidases) and their combinations, lowering the extraction temperature or ultrasonication increased the yield further. In addition, red-coloured low-molecular fraction (with similar absorption characteristics to R-phycoerythrin) was separated. For allophycocyanin phosphate pH 6 buffer gave slightly higher yield (0.09% using fluorescence detector and 0.12% using photodiode array detector), compared to citrate or acetate buffer at the same pH. However, the tested enzyme combination, temperature variation and ultrasonication were not so advantageous for allophyocyanin recovery. The phycocyanin concentration from the red algae was below the limit of detection.

Detection of Ochratoxin A From Poultry Feed Using High Pressure Liquid Chromatography

One hundred poultry feed samples comprising of commercially produced poultry feed (48) and poultry feed compounded by farmers (privately milled) (52) were collected from farms located in Minna and analysed for Ochratoxin A (OTA), a member of group 2 possible carcinogen by High Pressure Liquid Chromatographic (HPLC) method. Thirty seven percent of the commercial poultry feeds were contaminated with OTA at a range of 0 -236.73ug/kg while hundred percent of privately made feed were contaminated with OTA at a range of 22.76-226.5lug/kg. The finding of this investigation showed that 71% (71/100) of the sampled poultry feed has OTA concentrations which was far in excess of the maximum permissible limit of 5ug/kg (the European Union Standard). This is of serious health concern to the birds and humans that consume the poultry products.

Selecting an HPLC method for chemotaxonomic analysis of phytoplankton community in Mediterranean coastal lagoons

1 - Phytoplankton observations are commonly used to contribute to the assessment of aquatic ecosystem health and their trophic status. Compared to other methods, chemotaxonomic analysis based on High Pressure Liquid Chromatography (HPLC) presents many advantages (e.g., rapidity, reproducibility, and capacity to include pigments from all cell sizes), but its use in coastal lagoons studies is still not very common. The method of Wright et al., (1991) recommended by the UNESCO (Jeffrey et al., 1997) and most frequently used for phytoplankton analysis in coastal lagoons, so far, was selected and compared to the more novel method of Hagerthey et al. (2006). 2 - The two methods that differed slightly with respect to their solvent gradients during chromatography (mobile phase) and column (stationary phase), were tested using a pigment mix from DHI Water and Environment comprising 30 different pigments. Extraction methods were tested using replicates of 1l of sub-surface water from the Thau lagoon (South of France), sampled in June 2013. Optimization of the extraction was performed by testing different volumes of solvent (2 to 5 ml), different solvents based on a mix of methanol, acetone, dimethylformamide, water, compared to acetone 90% and pure methanol, as well as different extraction times (10 min to 2 h), and the addition of the ion-pairing agent tetrabutyl ammonium acetate hydroxide (TBAA). 3 - The second method of analysis allowed better separation and resolution of most of the pigments, especially of lutein and zeaxanthin. The early-eluting most polar pigments and the more hydrophobic pigments eluting in the end of the chromatogram (chlorophylls and carotenoids) showed also better separation and peak shapes. 5 mL of the mix of acetone/ methanol/ water (45:45:10) allowed the best extraction of the pigments. The use of TBAA showed negative effects. 4 - For pigment analysis in coastal lagoon, our final protocol used 1 h extraction with 5 mL of acetone/ methanol/ water, and analysis with the gradient from Hagerthey et al. (2006). On our analytical equipment it needed some adjustments. It uses a longer chromatography run and quantified the phytoplankton pigment markers better than the method of Wright et al. (1991).

ANTIOXIDANT AND ANTICANCER ACTIVITY OF EXTRACT AND FRACTIONS OBTAINED FROM DIOSPYROS MELANOXYLON ROXB. LEAVES AND CORRELATION WITH THEIR POLYPHENOLIC PROFILES

Objective: The current study evaluates the antioxidant and cytotoxic prospective of the leaves from Diospyros melanoxylon Roxb. (D. melanoxylon).Methods: Qualitative phytochemical analysis of the samples from D. melanoxylon was carried out for the detection of secondary metabolites. Total content of flavonoids, phenolics, triterpenoids, and tannins in D. melanoxylon was evaluated using colorimetric assay. Qualitative analysis of polyphenolic compounds was performed using HPLC method. The antioxidant activity was examined by assessing the various free radical scavenging assays. Five human cancerous cell lines (HeLa, MCF-7, HCT-116, PC-3, and HEK293), one rat colon cancer cell line (RCC 45) and two normal cell lines (NKE and WI-38) were used for evaluating the anticancer activity through methylene blue assay.Results: The results indicated that ethyl acetate fraction (EAF) and aqueous fraction (AQF) exhibited the remarkable content of flavonoids, phenolics, triterpenoids, and tannins. Both EAF and AQF have cytotoxic activity with significant IC50 values but not showed significant antioxidant activity. However, the most powerful anticancer activity was recorded by EAF followed by AQF in a dose-dependent manner. It was also revealed that EAF and AQF were biocompatible with the normal cell lines.Conclusion: Our principal finding exhibited a potent anticancer activity. Furthermore, it was also indicated that samples contain secondary metabolites like flavonoids, phenolics, triterpenoids, and tannins. We suggest that the further investigation on this herb is effective against various types of ailments including cancer.Keywords: Diospyros melanoxylon Roxb., Scanning Electron Microscopy (SEM), High-Pressure Liquid Chromatography (HPLC), Polyphenols, Antioxidant, Anticancer

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR THE DETERMINATION OF ALVIMOPAN IN RAT PLASMA

Objective: The present investigation demonstrates a simple, sensitive and accurate high pressure liquid chromatographic (HPLC) method for the determination of alvimopan (AMP) in rat plasma.Methods: The chromatographic separation was achieved within 10 min by using acetonitrile: potassium dihydrogen phosphate buffer pH 3.0 adjusted with orthophosphoric acid (50:50) as mobile phase on Altima Grace Smart C-18 column (5μ; 250 × 4.6 mm) at a flow rate of 1.0 ml/min with injection volume 50 µl. The drug was extracted from plasma by liquid-liquid extraction using a mixture of methanol: acetonitrile (50:50) as a solvent. The retention times of drug and internal standard were found to be 5.17 and 6.74 min, respectively. This method was validated as per the United States Food and Drug Administration (US-FDA) guidelines.Results: The results of the validation parameters were found to be within the acceptance limits. The method was linear in the concentration range from 5-1000 ng/ml (r2= 0.9998), and the extraction recovery was found to be 78.71±3.86% for AMP. The lower limit of quantification was found to be 5ng/ml, and the stability of recovered samples at different conditions was found to be more than 95%.Conclusion: The developed method possess good selectivity, specificity, there was no interference found in the plasma blanks at retention times of AMP and Internal Standard (IS). We found a good correlation between the peak area and concentration of the drug under prescribed conditions. Furthermore, the method can also be used to estimate the pharmacokinetic parameters of AMP.Keywords: Alvimopan, Liquid-liquid extraction, Method development, Matrix effect, Plasma, Recovery, Stability, Validation

Quantification of THC in Cannabis plants by fast-HPLC-DAD: A promising method for routine analyses

Nowadays, Gas Chromatography Mass Spectrometry (GC-MS) is mainly used in forensic sciences but suffers from limitations when the analysed compounds are thermally instable as it is the case for THC-A (Tetrahydrocannabinolic Acid) which is converted into Δ9-THC (Δ9-Tetrahydrocannabinol) that subsequently partially degrades. We propose herein a Fast High Pressure Liquid Chromatography (Fast-HPLC-DAD) method which allows the efficient separation of CBN (Cannabinol), CBD (Cannabidiol), THC-A and Δ9-THC, the major cannabinoids compounds found in cannabis plants in less than 5 min. Our method allows also the proper quantification of Δ9-THC in plant extracts using an external calibration method with a very good accuracy as pointed out by a recovery of 100.53 ± 3.12%. It is also an interesting low cost alternative to Ultra High Pressure Liquid Chromatography (UPLC) for routine analyses in forensic sciences.

A new cause of false positive voriconazole levels: Watch your collection tubes!

We communicate the interference of a compound of the blood collection tube with the accuracy of a validated high-pressure liquid chromatography method with ultraviolet detection for quantifying voriconazole levels, which led to false positive results. This could have serious consequences for patient management. Our advice is to implement external assessment programs.

Solubility Measurement and Thermodynamic Modeling of Benzoic Acid in Monosolvents and Binary Mixtures

The solid–liquid equilibrium of benzoic acid has been determined in six monosolvents—tributyl phosphate, diacetone alcohol, methyl-n-propyl ketone, methyl acetate, amyl acetate, and isooctane—at temperatures from 283.15 to 328.15 K and five binary systems—ethanol + hexane (288.15–328.15 K), isopropyl alcohol + hexane and chloroform + hexane (288.15–323.15 K), acetone + hexane (288.15–318.15 K), and acetone + water (288.15–318.15 K)—at atmospheric pressure with varying mole fraction of the binary mixture. The solubility was estimated by three different methods—titrimetry, gravimetry, and high-pressure liquid chromatography methods. The solubility of benzoic acid was found to increase with an increase in temperature and mole fraction. Experimental solubility data was correlated with various thermodynamic models such as the Buchowski equation, the NRTL model, and the modified Apelblat–Jouyban–Acree model. The mathematical models demonstrated that the calculated and experimental solubilities of benzoic acid i...

DEVELOPMENT AND VALIDATION OF HPLC METHOD FOR THE IGURATIMOD

Analytical method with accuracy, precision and stability indicting was developed for Iguratimod, achieved the required chromatographic separation by High pressure liquid chromatography (HPLC) techniqu e. HPLC method with accuracy, precision, robustness and stability indicating was developed and optimized for quantitative evaluation of Iguratimod as active pharmaceutical ingredient. Chromatographic separation for Iguratimod and its related compounds was achieved by using analytical column with stationary phase Inertsil ODS-3 (Dimension: length 150mm, internal diameter 4.6mm and particle size 5µ) and mobile phase (Buffer and acetonitrile in gradient proportion) at flow rate 1.0ml/minute with detection at 257nm. Squared correlation coefficient (r2) was observed more than 0.999 with concentration window of 12.5 to 75µg/ml. Iguratimod was retained at about 19.0±2.0 minutes. Forced degradation study for Iguratimod was carried out under the stress condition to get required degradation by using acid, base, thermal, photolytic and oxidation by hydrogen peroxide. Degradation products which are generated during stress condition did not interfere to active ingredient, Iguratimod. This analytical method optimized, validated for all the parameters specified in ICH guideline, to get results selective, accurate and precise in presence of degradation impurities.

HPLC Method for Ethylenethiourea in Biological and Environmental Samples

Objective. In view of both the economic importance of ethylenebisdithiocarbamate (EBDC) fungicides in the current agricultural practice and the potential health hazards associated with ethylenethiourea (ETU) exposure, this study aimed to develop and validate a high-pressure liquid chromatography (HPLC) method to determine ETU in biological and environmental samples.
 Methods. The samples were pre-treated according to sample types and were analyzed for ETU using a reversedphase HPLC system (JASCOᴿ) equipped with UV detector set at 230 nm using C18 bonded silica column and a mobile phase of 0.05M ammonium acetate in methanol (95:5).
 Results. The method showed a limit of detection of 0.2 ug/L, with a precision of 3.33 to 12.86 %CV and accuracy of >90% at 1, 10 and 100 ug/L of ETU in all sample types. The calibration curve was linear from 1 to 200 ug/L for blood, air and water samples and 1 to 2000 ug/L for urine.
 Conclusion. This method showed an acceptable accuracy, precision, sensitivity and specificity and was used subsequently to determine ETU levels in blood, urine, air, soil and water samples among banana plantation workers.

ENANTIOSELECTIVE ANALYSIS OF GUAIFENESIN IN BULK AND PHARMACEUTICAL DOSAGE FORMS BY CHIRAL REVERSE PHASE HPLC-PDA METHOD

A novel, accurate and precise chiral reverse-phase high pressure liquid chromatographic method was developed for enantioselective analysis of guaifenesin (GFN) in bulk and tablet dosage forms. Chiral separation was achieved on phenomenex Lux Cellulose-4 column (250×4.6mm, 5μ) using 0.02% formic acid: acetonitrile (90:10 V/V) as the mobile phase at a flow rate of 1mL/min at 230nm. The retention times of GFN enantiomers A and B was 15 and 16 minutes, respectively, with good peak resolutions and showing good linearity in the concentration range of 10-50 μg/mL (R2 > 0.999). The developed method was validated as per the International Conference on Harmonization guidelines and the results were well within the acceptable limits. The percentage assay in tablet dosage form was found to be 98.8 and 98.2 respectively, for enantiomers A and B and was with in the compendial specifications, demonstrating the suitability of developed method for enantioselective analysis of guaifenesin racemic mixture.

Complexation of diclofenac sodium with hydroxy propyl betacyclodextrin improves its solubility and stability in ampoule solution which is determined by HPLC

Diclofenac sodium injection is widely used in medicine as antinflamatory and antirheumatic agent with a therapeutic value of 25 mg / ml. However, diclofenac sodium is poorly soluble which forms a problem in its manufacturing as an injection. The available commercial products of diclofenac sodium ampoules have used different types of solubilizing agents as benzyl alcohol which is an irritant in a concentration more than 3% while the other manufacturers used propylene glycol which has toxic impurities. In this present work, Diclofenac Sodium injection is prepared by using Hydroxy Propyl Beta Cyclodextrine ; a natural and safe excipient in formulation of ampoule solution which formed an inclusion complex compounds with Diclofenac Sodium ,render it very soluble and more stable. The finished product of ampoules were subjected to the stability study by storing the samples at 40°C and 75% RH for six months and the physico-chemical properties of the samples were tested at different periods. The results showed no change in appearance of the ampoules solution along the study time .In addition a reversed –phase high pressure liquid chromatographic method was developed and applied in studying the behavior of diclofenac Sodium in solution and its resistance to the high temperature challenger. The developed HPLC method was proved to be accurate and able to detect the degradation products of Diclofenac Sodium in solution.

Bacterial Cell Wall Precursor Phosphatase Assays Using Thin-layer Chromatography (TLC) and High Pressure Liquid Chromatography (HPLC).

Peptidoglycan encases the bacterial cytoplasmic membrane to protect the cell from lysis due to the turgor. The final steps of peptidoglycan synthesis require a membrane-anchored substrate called lipid II, in which the peptidoglycan subunit is linked to the carrier lipid undecaprenol via a pyrophosphate moiety. Lipid II is the target of glycopeptide antibiotics and several antimicrobial peptides, and is degraded by 'attacking' enzymes involved in bacterial competition to induce lysis. Here we describe two protocols using thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC), respectively, to assay the digestion of lipid II by phosphatases such as Colicin M or the LXG toxin protein TelC from Streptococcus intermedius. The TLC method can also monitor the digestion of undecaprenyl (pyro)phosphate, whereas the HPLC method allows to separate the di-, mono- or unphosphorylated disaccharide pentapeptide products of lipid II.