Chromatographic detection of residual cellular DNA on short monolithic columns

Analysis of crude samples from biotechnological processes is often required to demonstrate that residual host cell impurities are reduced or eliminated during purification. Current knowledge suggests that a continuous-cell-line DNA can be considered a cellular contaminant rather than a significant risk factor requiring removal to extremely low levels. Anion-exchange chromatography is one of the most important methods used in the downstream processing and analysis of different biomolecules. In this article, an application using Convective Interaction Media monolithic columns to improve the detection of residual cellular DNA is described.     

Purification of chemically synthesised dinucleoside(5′,5′) polyphosphates by displacement chromatography

Dinucleoside(5′,5′) polyphosphates (Ap_nA, Ap_nG, Gp_nG, n=3–6) are new group of hormones controlling important biological processes. Because some of the dinucleoside(5′,5′) polyphosphates are commercially not available purification of chemical synthesised dinucleoside(5′,5′) polyphosphates became necessary in order to test their physiological and pharmacological properties. It was the aim of this study to find a method which allows purification of 0.1–0.2 g quantities of dinucleoside polyphosphates by analytical HPLC columns yielding products with impurities lower than 1.0%. Adenosine(5′)-polyphospho-(5′)guanosines were synthesised by mixing the corresponding mononucleotides. The reaction results in a complex mixture of Ap_nA, Ap_nG and Gp_nG (with n=3–6 in all cases). The reaction mixture was concentrated on a preparative C₁₈ reversed-phase column. The concentrate was displaced on a reversed-phase stationary. As a result of displacement chromatography, anion-exchange chromatography in gradient modus yielded baseline separated dinucleoside polyphosphates (homogeneity of the fractions>99%). The identity of the substances were determined by matrix assisted laser desorption ionisation mass spectrometry.     

Two-dimensional HPLC on-line analysis of phosphopeptides using titania and monolithic columns

Conventional and comprehensive two-dimensional (2D) HPLC systems using the combination of titania and monolithic columns were established for the on-line analysis of phosphopeptides. Compared with immobilized metal affinity chromatography of a general method for the analysis of phosphopeptides, the use of titania columns in the analysis permits the specific isolation of phosphopeptides in a higher yield. Using the current 2D HPLC systems, phosphopeptides were specifically isolated from nonphosphorylated peptides by the first-dimension titania column, followed by the high-speed separation of the phosphopeptides by the second-dimension monolithic column. Proteolytic digests of beta-casein were analyzed within 30 min using the comprehensive 2D HPLC system; all phosphopeptides from beta-casein could be efficiently isolated and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The comprehensive 2D HPLC system coupled with mass spectrometry will be useful for high-throughput and on-line phosphoproteome analyses.     

Separation and quantification of alkylphosphocholines by reversed phase high performance liquid chromatography

Alkylphosphocholines represent a new class of drugs with remarkable antineoplastic and antiprotozoal activity. For instance, hexadecylphosphocholine has been approved for the topical treatment of skin metastasis. In addition, it was successfully studied in India for the treatment of leishmaniasis. Different phase-I and phase-II-trials resulted in cure rates of more than 97%. To optimize antitumor or antiprotozoal activity, we have prepared alkylphosphocholines differing in chain length and unsaturation. For the qualitative and quantitative analysis of these longer chain analogues, we have used isocratic high performance liquid chromatography. The separation of the alkylphosphocholines with different chain lengths in this reversed phase HPLC system was achieved on a YMC-TMS column with a mobile phase consisting of methanol-water (85:15; v/v) at a flow rate of 1.0 ml/min. Furthermore the cis-/trans-isomers such as oleylphosphocholine and elaidylphosphocholine were clearly separated on a YMC-C8 column with a methanol-water mixture (80:20; v/v) as mobile phase. In the described reversed phase HPLC systems simple refractive index detection and UV detection allow the sensitive and quantitative determination of alkylphosphocholines. These methods are very important for reproducible identification and quantitative determination of saturated and mono-unsaturated alkylphosphocholines with alkyl residues containing up to 25 carbon atoms.     

The use of a monolithic column to improve the simultaneous determination of four cephalosporin antibiotics in pharmaceuticals and body fluids by HPLC after solid phase extraction--a comparison with a conventional reversed-phase silica-based column

The use of a monolithic column (Chromolith, SpeedROD RP-18e, by Merck) was studied on the determination of cephalosporin antibiotics. Results were compared with those from a previously developed analytical method using conventional silica-based analytical column. A rapid, accurate and sensitive method has been developed and validated for the quantitative simultaneous determination of four cephalosporins: Cephalexine and Cephadroxil (first generation), Cefaclor (second generation) and Cefotaxim (third generation) in pharmaceuticals as well as in human blood serum and urine. Hydroflumethiazide (HFM) (3,4-dihydro-6(trifluoromethyl)-2H-1,2,4-benzothiadiazine-7-sulfonamide-1,1-dioxide) was used as an internal standard at a concentration of 1.5 ng/microL. A rectilinear relationship was observed up to 5 ng/microL for the four compounds. Analysis time was less than 4 min. The statistical evaluation of the method was examined by means of within-day repeatability (n=8) and day-to-day precision (n=8) and was found to be satisfactory with high accuracy and precision results. The method was applied to the determination of the cephalosporins in commercial pharmaceuticals and in biological fluids: human blood serum after solid phase extraction and urine simply after filtration and dilution. Recovery of analytes in spiked serum samples was in the range from 88.7 to 107.8%, while for urine samples recovery was from 98.0 to 105.6%. By comparing the figures of merit for the monolithic column and the silica-based one, regarding the determination of the four cephalosporins investigated in the present study, the outstanding efficiency of the monolithic column can be noticed.     

Determination of terbutaline enantiomers in biological samples using liquid chromatography with coupled columns

The purpose of this work was to develop and validate a method for the separation and determination of the enantiomers of terbutaline in plasma and intestinal juice. Terbutaline was extracted from plasma and intestinal juice by liquid-solid extraction on small C18 cartridges. The extract was then analyzed by coupled column liquid chromatography with amperometric detection. For chiral separation a beta-cyclodextrin phase was used. The within-day variation (Cv) on spiked plasma samples was in the range 0.8-6.4% at 3.8-33.8 nmol/liter for the (-)-enantiomer, and 2.6-23.0% at 1.3-11.3 nmol/liter for the (+)-enantiomer. The between-day variation on spiked plasma samples was 5.5% at 10.7 nmol/liter and 13.6% at 4.3 nmol/liter for the (-)-and (+)-enantiomers, respectively. The within-day variation for intestinal juice was in the range 0.7-1.5% at 5.6-30.0 mumol/liter for the (+)-enantiomer.     

Determination of propiomazine in rat plasma by direct injection on coupled liquid chromatography columns with electrochemical detection

This method describes the determination of propiomazine by direct injection of rat plasma into a chromatography system based on coupled reversed-phase columns. An extraction column, packed with porous silica particles with covalent-bound ₁-acid glycoprotein (AGP), was used to separate the plasma proteins from the analyte. After isolation the analyte was transferred to the analytical column for separation and detection. Propiomazine was detected by an electrochemical detector and the limit of quantification was 2.0 ng/ml (100 pg injected). The absolute recovery was 80.9±2.4% at 9.0 ng/ml level. The inter-day and intra-day precision was 10.9% (5.6 ng/ml) and 2.8% (9.0 ng/ml), respectively.     

Simultaneous quantification of free nucleotides in complex biological samples using ion pair reversed phase liquid chromatography isotope dilution tandem mass spectrometry

A new sensitive and accurate analytical method has been developed for quantification of intracellular nucleotides in complex biological samples from cultured cells of different microorganisms such as Saccharomyces cerevisiae, Escherichia coli, and Penicillium chrysogenum. This method is based on ion pair reversed phase liquid chromatography electrospray ionization isotope dilution tandem mass spectrometry (IP-LC-ESI-ID-MS/MS. A good separation and low detection limits were observed for these compounds using dibutylamine as volatile ion pair reagent in the mobile phase of the LC. Uniformly ¹³C-labeled isotopes of nucleotides were used as internal standards for both extraction and quantification of intracellular nucleotides. The method was validated by determining the linearity, sensitivity, and repeatability.     

Preparation of highly efficient monolithic silica capillary columns for the separations in weak cation-exchange and HILIC modes

Weak cation-exchange (WCX) and HILIC modes columns were prepared by on-column polymerization of acrylic acid on monolithic silica capillary columns modified with N-(3-triethoxysilylpropyl)methacrylamide anchor groups. The polymer-coated columns could be used for HILIC mode separation of pyridylamino (PA)-sugars and peptides including a tryptic digest of BSA, while for weak cation-exchange mode for the separation of proteins and nucleosides even at high linear velocity. The poly(acrylic acid) coated monolithic silica capillary columns showed greater retention toward PA-sugars than a polyacrylamide coated monolithic silica capillary columns prepared in the same manner. Proteins and nucleosides were separated effectively at pH 6.9 using the same column. The column provided fair permeability after the polymer-coating step. High-speed separation of proteins at u=4.66 mm/s with high efficiency was shown to be possible, while high-speed separation of nucleosides has achieved within one minute using the column at u=8.67 mm/s, suggesting that the column will be suitable for the second dimension separation of multidimensional HPLC systems.     

大孔树脂吸附与反相色谱纯化恩拉霉素

恩拉霉素作为多肽类抗生素,是一种新型、安全的饲料添加剂。本文建立了一条基于大孔树脂初纯和反相色谱精制的分离纯化工艺。该工艺路线首先使用AB-8大孔树脂在0.012 mol/L盐酸溶液-甲醇(50:50,V/V)缓冲液条件下洗脱实现恩拉霉素初步纯化,再使用制备型C18反相色谱柱在0.05 mol/L磷酸二氢钠-乙腈(70:30,V/V)(p H 4.5)缓冲液洗脱下实现恩拉霉素a和b的有效分离,a、b两个组分纯度分别达到98.5%和98.0%,a和b两种有效成分的总收率为29.2%。本研究为恩拉霉素a和b两种纯品的制备以及高纯度恩拉霉素产品的生产提供了参考。     

Separation and quantification of neoagaro- and agaro-oligosaccharide products generated from agarose digestion by beta-agarase and HCl in liquid chromatography systems

A series of neoagaro-oligosaccharides (NAOS) were separated and isolated by beta-agarase digestion and agaro-oligosaccharides (AOS) by HCl hydrolysis from agarose with defined quantity and degree of polymerization (DP). Profiles of the oligomer length in the crude product mixtures were monitored by two high-performance liquid chromatography (HPLC) systems: size-exclusion chromatography (SEC) and NH2-column chromatography (NH2-HPLC), coupled with an evaporative light-scattering detector (ELSD). Calibration curves were established separately to identify the DP and quantify the amount of the oligomer products analyzed in the two systems. Each system was optimized to generate a spectrum of saccharide oligomers with various DP, where the reaction yield for NAOS was 52.7% by 4U/mg beta-agarase and for AOS was 45.6% by 0.4M HCl. SEC resolved the product in size ranges consisting of DP 1-22 for NAOS and DP 1-14 for AOS. NH2-HPLC clearly resolved both distinct saccharide product sizes within DP 12. The optimized system was connected with a fraction collector to isolate and quantify these individually separated products. The total product yields of the recovered NAOS of DP 1-22 and AOS of DP 1-14 by the SEC system were 84.7% and 82.9%, respectively. NH2-HPLC recovered NAOS and AOS, both with a DP of 1-10 with total product yields of 48.9% and 90.0%, respectively. Isolated NAOS and AOS product fractions were inspected by (1)H NMR spectroscopy and ESIMS spectrometry to confirm structure, molecular mass, and purity. This study established feasible systems for the preparation and qualitative and quantitative measurements, as well as for the isolation of various sizes of oligomers generated from agarose.     

Rapid identification and quantification of Collinsella aerofaciens using PCR

The number and incidence of Collinsella aerofaciens in the human intestine are the highest among Gram-positive non-spore-forming bacilli. Identification of this species is very difficult and requires considerable time. A PCR-based identification system using C. aerofaciens-specific primers is described. Using this PCR method, we identified 181 C. aerofaciens-like species isolated from human feces. These 181 strains were identified using the traditional method in past studies. Results of both methods matched. The direct detection method was performed using human feces samples from seven adults. Nested PCR was applied directly to the samples and all seven samples were positive. Quantification studies were performed using LightCycler?trade mark omitted?. The assay uses a double-stranded DNA dye to continuously monitor product formation and in a short time is able to quantify samples to 5 log units in concentration.     

Phosphates of riboflavin and riboflavin analogs: A reinvestigation by high-performance liquid chromatography

Phosphoric acid esters of riboflavin can be easily separated by reverse-phase high-performance liquid chromatography using eluants of 0.1 M ammonium formate in aqueous methanol. Commercial FMN preparations contained seven different flavin phosphates; the content of riboflavin 5'-phosphate was 70-75% and is in agreement with previous studies. Millimole amounts of crude FMN can be processed by preparative HPLC. The method permits the preparation of greater than 99%-pure 5'-FMN. The following compounds were isolated in pure form and their structures determined: riboflavin 4'-phosphate, riboflavin 3'-phosphate, riboflavin 4',5'-diphosphate; riboflavin 3',4'-diphosphate, and riboflavin 3',5'-diphosphate. The latter compound binds tightly to apoflavodoxin from Megasphaera elsdenii (KD = 9.7 X 10(-9) M). The bound flavin has high catalytic activity, thus representing a novel type of FMN analog. A wide variety of structural analogs of FMN can be obtained in pure form by preparative HPLC.     

Molecular quantification of environmental DNA using microfluidics and digital PCR

Real-time PCR has been widely used to evaluate gene abundance in natural microbial habitats. However, PCR-inhibitory substances often reduce the efficiency of PCR, leading to the underestimation of target gene copy numbers. Digital PCR using microfluidics is a new approach that allows absolute quantification of DNA molecules. In this study, digital PCR was applied to environmental samples, and the effect of PCR inhibitors on DNA quantification was tested. In the control experiment using λ DNA and humic acids, underestimation of λ DNA at 1/4400 of the theoretical value was observed with 6.58ngμL(-1) humic acids. In contrast, digital PCR provided accurate quantification data with a concentration of humic acids up to 9.34ngμL(-1). The inhibitory effect of paddy field soil extract on quantification of the archaeal 16S rRNA gene was also tested. By diluting the DNA extract, quantified copy numbers from real-time PCR and digital PCR became similar, indicating that dilution was a useful way to remedy PCR inhibition. The dilution strategy was, however, not applicable to all natural environmental samples. For example, when marine subsurface sediment samples were tested the copy number of archaeal 16S rRNA genes was 1.04×10(3)copies/g-sediment by digital PCR, whereas real-time PCR only resulted in 4.64×10(2)copies/g-sediment, which was most likely due to an inhibitory effect. The data from this study demonstrated that inhibitory substances had little effect on DNA quantification using microfluidics and digital PCR, and showed the great advantages of digital PCR in accurate quantifications of DNA extracted from various microbial habitats.     

Enantiomeric separation of norgestrel by reversed phase high-performance liquid chromatography using eluents containing hydroxypropyl-beta-cyclodextrin in stereoselective skin permeation study

The chiral separation of norgestrel enantiomers using reversed-phase high-performance liquid chromatography (RP-HPLC) was studied with hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as chiral mobile phase additive. The effect of mobile phase composition, concentration of HP-beta-CD and column temperature on enantioselective separation were investigated. The quantification properties of the developed RP-HPLC method were examined. A baseline separation of norgestrel enantiomers was achieved on a Agilent ZORBAX Eclipse XDB-C8 column (150 mm x 4.6 mm i.d., 5 microm). The mobile phase was a mixture of acetonitrile and phosphate buffer (pH 5.0, 20 mM) containing 25 mM HP-beta-CD (30:70, v/v) with a flow rate of 1.0 ml/min. The UV detector was set at 240 nm. Calibration curves were linear (n=8) in the range of 0.2-25 microg/ml, the limit of detection and quantitation were 0.10 and 0.20 microg/ml, respectively, for racemic norgestrel. The values of RSD of repeatability and intermediate precision for spiked sample were less than 4.8%. The method was successfully applied to the enantioselective determination of this drug in stereoselective skin permeation study.     

Determination of stereo- and positional isomers of oxygenated triterpenoids by reversed phase high performance liquid chromatography

Twenty four oxygenated triterpenoids, including eight pairs of stereoisomers and five pairs of positional isomers, could be separated by reversed phase HPLC. The capacity factors obtained in methanol-water and acetonitrile-water solvent systems made it possible to correlate the molecular polarities due to the presence of multiple oxygenated functional groups in these compounds. It was found that the number and position of functional groups as well as the stereochemistry of these functional groups played important roles in governing the polarity of these lanostanoid acids. The polarity weighting factors were in the following order: 3 beta-OH greater than 3 alpha-OH greater than 3 alpha-OAc greater than 3 beta-OAc. The contribution to polarity due to 15 alpha-OAc and 22 beta-OAc was probably very similar. The unique stereochemical character and eluting sequences of the lanostanoid acids provide information to generate empirical rules for predicting the role of individual polar functional groups in the chromatographic behavior in reversed phase HPLC.     

Enantiomeric analysis of amino acids by using comprehensive two-dimensional gas chromatography

The chiral separation of amino acids (AA) derivatised with ethyl chloroformate by using comprehensive two-dimensional gas chromatography is reported. A commercially available enantioselective capillary column (Chirasil-l-Val) has been tested as first-dimension column. Two nonenantioselective stationary phases (BPX50 and BP1) with different column lengths were combined with the enantioselective column, which represent chiral/polar and chiral/low-polarity column sets, respectively. These column sets were evaluated to determine the most useful column combination to provide improved separation efficiency of enantioselective AA analysis. Separations of AA mixtures derivatised either as their N-trifluoroacetyl methyl esters or with methyl chloroformate, performed on a chiral/low-polarity column set, are also shown. The method was demonstrated for chiral analysis of AAs in different beer samples. The major AA in the beer samples was proline with amounts ranging from around 65-95% with minor contents of glycine and the l-enantiomers of alanine, valine, leucine, and isoleucine. Small amounts of d-alanine, at about 1, 1.5, and 15% were detected in the three samples.     

A method to determine tannin concentration by the measurement and quantification of protein — tannin interactions

Summary Protein determinations on protein-tannin complexes after protein isolation (gel filtration and trichloroacetic acid [TCA] precipitation) or phenolic extraction (polyvinyl pyrrolidone [PVP] and organic solvent precipitation) were unsuccessuful. Kjeldahl determinations of the amount of unprecipitated protein bovine serum albumin [BSA] showed a sigmoid relationship with increasing concentrations of tannins. A similar relationship was found for the reduced viscosity of BSA and plant protein, and the concentration of tannin. Non-linear regression and curve normalization allowed three variable (k ₁, k ₂ and T _1/2) to be defined for the quantification of the protein-tannin interaction/s. Such a treatment may be useful in studies of the role of tannins in plant-herbivore interactions.     

Liquid- and gas-phase nitration of bovine serum albumin studied by LC-MS and LC-MS/MS using monolithic columns

Post-translational nitration of proteins was analyzed by capillary reversed-phase high-performance liquid chromatography (RP-HPLC) on-line interfaced to electrospray ionization mass spectrometry (ESI--MS) or tandem mass spectrometry (ESI--MS/MS). Both methods were compared using a tryptic digest of bovine serum albumin (BSA) and yielded sequence coverages of 95% and 33% with RP-HPLC--ESI--MS and RP-HPLC--ESI--MS/MS, respectively. At least 95% of the tyrosines were covered by the former method, whereas the latter method only detected less than 50% of the tyrosine-containing peptides. Upon liquid-phase nitration of BSA in aqueous solution using an excess of tetranitromethane, at least 16 of the 20 tyrosine residues were found to be nitrated. After exposure of solid BSA samples to gaseous nitrogen dioxide and ozone at atmospherically relevant concentration levels, only 3 nitrated peptides were detected. By use of such a model system, RP-HPLC--ESI--MS proved to be a rapid and highly efficient method for the comprehensive and quantitative detection of protein nitration.