Desalting of nucleotides by reverse-phase high-performance liquid chromatography

Chromatography of AMP, NAD⁺, or NADH on a reverse-phase C₁₈ Porasil B column rapidly removes ammonium formate or potassium phosphate from 90% of the nucleotide. Earlier reports showed these salts could not be separated from nucleotides by conventional desalting using gel filtration.     

The analysis of acyl-coenzyme A derivatives by reverse-phase high-performance liquid chromatography

A method for determining tissue levels of Coenzyme A and various short-chain-length acyl-CoA derivatives using high-performance liquid chromatography is presented. Separation of the various compounds was accomplished using a reverse-phase Spherisorb ODS II, 5-microns C18 column. Mobile-phase solvents were (a) potassium phosphate, 220 mM; thiodiglycol (2,2-thiodiethanol), 0.05% (v/v), pH 4.0 and (b) methanol, 98%; chloroform; 2% (v/v). The various acyl-CoA derivatives were detected by monitoring the column effluent at 254 nm. Nearly baseline separation was obtained for a standard mixture of free CoASH, methylmalonyl-CoA, beta-hydroxy-beta-methylglutaryl-CoA, succinyl-CoA, acetoacetyl-CoA, acetyl-CoA, propionyl-CoA, isobutyryl-CoA, beta-methyl-crotonyl-CoA, and isovaleryl-CoA. CoA derivative profiles were determined in neutralized perchloric acid extracts of perfused rat hearts and livers and of isolated rat liver mitochondria to demonstrate the utility of this method for assessing the levels of CoA derivatives in biological samples.     

Simultaneous glycosylation analysis of human serum glycoproteins by high-performance liquid chromatography/tandem mass spectrometry

Changes in the glycosylation of some serum proteins are associated with certain diseases. In this study, we performed simultaneous site-specific glycosylation analysis of abundant serum glycoproteins by LC/Qq-TOF MS of human serum tryptic digest, the albumin of which was depleted. The glycopeptide peaks on the chromatogram were basically assigned by database searching with modified peak-list text files of MS/MS spectra and then based on mass differences of glycan units from characterized glycopeptides. Glycopeptide of IgG, haptoglobin and ceruloplasmin were confirmed by means of a comparison of their retention times and m/z values with those obtained by LC/MS of commercially available glycoproteins. Mass spectrometric carbohydrate heterogeneity in the assigned glycopeptides was analyzed by an additional LC/MS. We successfully demonstrated site-specific glycosylation of 23 sites in abundant serum glycoproteins.     

Determination of ADP-ribosyl arginine anomers by reverse-phase high-performance liquid chromatography

A simple and rapid method for the determination of ADP-ribosyl arginine anomers was devised. Analysis is performed by reverse-phase high-performance liquid chromatography on a 5-micron Cosmosil 5C18 column and uv detection. ADP-ribosylation of arginine by hen liver ADP-ribosyl-transferase and the effect of pH on anomerization are also presented.     

Isolation of spectrin subunits by reverse-phase high-performance liquid chromatography.

We present a one-step uncomplicated method of separation of spectrin subunits. The method is based on reverse-phase HPLC employing an analytical C4 column. Reverse-phase HPLC combines the steps of dissociation and separation of spectrin subunits. The method can be applied to different spectrin isoforms. It can be used for analytical purposes, as well as for small-scale (<0.4 mg) isolation of spectrin subunits.     

Analysis of dansyl amino acids by reverse-phase high-performance liquid chromatography

All 24 dansyl amino acids were separated by reverse-phase high-performance liquid chromatography on Develosil C8-5, using a linear gradient made from Tris-HCl buffer (pH 7.75) and methanol. A linear relationship between the amount of sample and peak area was found over the range of 6 to 300 ng (0.02–1 nmol) of dansyl derivatives. An application of this method to the NH₂-terminal analysis of lysozyme is described.     

Assay for ADP-ribosyl cyclase by reverse-phase high-performance liquid chromatography.

Cyclic ADP-ribose (cADPR), a natural metabolite of beta-NAD(+), is a second messenger for Ca(2+) signaling in T cells. As a tool for purification and identification of ADP-ribosyl cyclase(s) in T cells, a sensitive and specific enzymatic assay using 1,N(6)-etheno-NAD(+) as substrate was developed. A major problem-the sensitivity of 1,N(6)-etheno-cADPR toward the extraction medium perchloric acid-was solved by replacing the perchloric acid extraction procedure of nucleotides by a filtration step. Standard compounds for the HPLC analysis of ADP-ribosyl cyclases and NAD(+)-glycohydrolases, e.g., 1,N(6)-etheno-cADPR, 1,N(6)-etheno-ADPR, and 1,N(6)-etheno-AMP, were produced by ADP-ribosyl cyclase from Aplysia californica and dinucleotide pyrophosphatase. The assay was applied to subcellular fractions prepared from human Jurkat T cells. As a result ADP-ribosyl cyclase and NAD(+)-glycohydrolase activity could be detected and precisely quantified in different subcellular fractions indicating the presence of different isoenzymes in T cells.     

Quantitative analysis of human serum corticosterone by high-performance liquid chromatography coupled to electrospray ionization mass spectrometry

An original method based upon high-performance liquid chromatography coupled to electrospray ionization mass spectrometry has been developed for corticosterone (B) quantification in human serum. After extraction by diethyl ether using triamcinolone (T) as an internal standard, solutes are separated on a C₁₈ microbore column (250×1.0 mm, I.D.), using acetonitrile–water–formic acid (40:59.9:0.1, v/v/v) as the mobile phase (flow-rate 40 μl/min). Detection is performed on an API 1 single quadrupole mass spectrometer equipped with a ESI interface and operated in positive ionization mode. Corticosterone quantifications were realized by computing peak area ratios (B/T) of the serum extracts analyzed in SIM mode (m/z 347 and m/z 395 for B and T, respectively), and comparing them with the calibration curve (r=0.998).     

Separation and purification of individual neurofilament proteins by reverse-phase high-performance liquid chromatography

A reverse-phase HPLC method was developed to separate individual neurofilament proteins (210,000, 160,000, and 70,000 Da) from the glial fibrillary acid protein. It is useful for analytical or preparative methods, with yields higher than 80%. The method represents improvement over previous methods in speed, efficiency, and purity. Combining this HPLC method with the conventional chromatographic method on DEAE-cellulose, highly purified individual neurofilament proteins can be obtained in large scale.     

Therapeutic drug monitoring of flecainide in serum using high-performance liquid chromatography and electrospray mass spectrometry

High-performance liquid chromatography with electrospray mass spectrometry (LC–MS) was used for analysis of the drug flecainide in serum. The clean-up was performed by solid-phase extraction, and an aromatic ring positional isomer was used as internal standard. Results from method validation on spiked serum samples showed excellent reproducibility; intra- and inter-assay variations (C.V.% and %Bias) were less than 6% within the therapeutic concentration range of the drug (0.2–1.0 μg/ml). Linearity was demonstrated from 0.05 to 2.0 μg/ml. The limit of detection and quantification was 0.025 and 0.05 μg/ml, respectively. Due to the high selectivity of the mass spectrometric detection, no interferences were observed. Results from clinical samples (n=18) from patients in treatment with Tambocor (flecainide acetate) showed excellent correlation with parallel data obtained from a method based on high-performance liquid chromatography (HPLC) with fluorescence detection after liquid/liquid extraction. The chromatographic separation of flecainide and internal standard was improved compared to earlier HPLC methods. The methodology is simple, accurate and requires only 0.25 ml of sample. It is a well suited method for routine therapeutic drug monitoring in a hospital or clinical chemistry laboratory.     

Assay of mouse liver uroporphyrinogen decarboxylase by reverse-phase high-performance liquid chromatography

A method for the estimation of hepatic uroporphyrinogen decarboxylase activity employing reverse-phase HPLC is described. Mouse liver homogenate in 0.25 M sucrose was pretreated with a suspension of cellulose phosphate and then centrifuged to remove hemoglobin and debris. The supernatant was used as the enzyme source. Incubations were acidified, oxidized, and centrifuged only before analysis of the porphyrins formed, using a Spherisorb ODS column and a gradient solvent system constructed from methanol/lithium citrate mixtures. Coproporphyrinogen formation by BALB/c mouse liver supernatant was estimated as about 5.0 and 9.1 pmol/min/mg protein from uroporphyrinogens I and III, respectively, at 10 microM substrate concentration and pH 6.8. Decarboxylation of pentacarboxyporphyrinogens (the last step in coproporphyrinogen formation) proved to be easily measured. Coproporphyrinogen formation from pentacarboxyporphyrinogen III abd (20 microM) at pH 6.8 was about 109 pmol/min/mg protein. Pentacarboxyporphyrinogen I was not as good a substrate as III abd but was decarboxylated faster at pH 5.4 than at 6.8, and at the lower pH and at 10 microM concentration of substrate 42 pmol of coproporphyrinogen was formed/min/mg protein. These results compared favorably with those obtained by previously published procedures involving time-consuming extraction and esterification steps.     

Separation of 5'-ribonucleoside monophosphates by ion-pair reverse-phase high-performance liquid chromatography

We have developed computer programs for characterization of ligand-binding systems in terms of continuous affinity distributions of arbitrary shape based on a numerical finite difference method. This method provides an excellent initial estimate of the affinity distribution, which can be further refined by means of nonlinear least-squares curve fitting. The method has been extensively tested for several cases including receptor heterogeneity, cooperativity, and for several examples of experimental design (e.g., ligand concentrations), and various levels of random and systematic experimental errors. The results provide a guide to experimental design, and indicate limits to the resolution obtained by ligand-binding studies, irrespective of the method of analysis.     

Supports for reverse-phase high-performance liquid chromatography of large proteins

Reverse-phase high-performance liquid chromatography supports have been developed for use in separating proteins up to 300,000 M_r. They are based on silica supports to which octyl, cyanopropyl, or diphenyl groups are covalently bonded. Their effectiveness in rapidly separating several standard proteins is demonstrated. Applications presented include the separation of the α₁ and α₂ chains of chick Type I collagen within 1 h and the separation of the α and β components of human Type I collagen.     

Urinary neopterin quantification by reverse-phase high-performance liquid chromatography with ultraviolet detection

Neopterin plays an important role in the malignant disease diagnostics. However, the methods employed in neopterin determination are generally difficult and/or time consuming. The aim of this work was to standardize a practical method to quantify neopterin using high-performance liquid chromatography-ultraviolet (HPLC-UV) and quantify it in patients with systemic lupus erythematosus (SLE). Urine was collected from healthy subjects (n= 49), patients with inactive (n= 15), active (n= 28), and highly active SLE (n= 6). The HPLC was performed using two coupled reverse-phase columns eluted with 150 mM sodium phosphate, pH 4.0, under a flow rate of 0.8 ml/min, with UV detector set at 353 nm and 100-fold diluted urines. The inter- and intra-assay studies presented an imprecision of 12.5% and 12.9% for quality controls of 3.94 and 1.1 micromol/ml, respectively. Recovery from 79.5% to 82% was observed throughout the assay's linear range. Subjects with active (874.2 +/- 165.38 micromol/mol creatinin) and highly active SLE (1753.8 +/- 453.9 micromol/mol creatinin) showed three- and sixfold increased neopterin levels, respectively, compared to subjects with inactive SLE (314.3 +/- 121.3 micromol/mol creatinin) and healthy subjects (294.6 +/- 178.6 micromol/mol creatinin) (P< 0.05). Briefly, the proposed method was precise, specific, and reproducible, not invasive and allows the urinary neopterin quantification only with UV detection.     

Determination of trimethoprim in bovine serum by high-performance liquid chromatography with confirmation by thermospray liquid chromatography—mass spectrometry

A high-performance liquid chromatographic (HPLC) method with a detection limit of 5 ng/ml was developed for the analysis of trimethoprim in bovine serum. Trimethoprim and the internal standard, ormetoprim, under alkaline conditions, were first extracted into dichloromethane and then back-extracted into dilute sulphuric acid (0.15 M) and cleaned-up on a C₁₈ cartridge. Trimethoprim was quantified on a C₁₈ column using a triethylammonium acetate—acetonitrile—methanol (16:3:1, v/v/v) mobile phase at a flow-rate of 1.5 ml/min, with ultraviolet detection at 225 nm. This method was used to verify the accuracy of test responses obtained with the Brilliant Black Reduction test, a rapid screening method, for trimethoprim levels in the serum of steers treated with Trivetrin. Confirmation of the presence of trimethoprim in the sample extract was obtained by thermospray HPLC—mass spectrometry.     

Analysis of oligosaccharides by on-line high-performance liquid chromatography and ion-spray mass spectrometry.

Oligosaccharides were analyzed by a combination of high-performance liquid chromatography (HPLC) and mass spectrometry (MS). First, oligosaccharides labeled with 2-aminopyridine were studied to see if they could be analyzed by MS under the conditions used for separation by HPLC. Pyridylamino (PA)-oligosaccharides could be analyzed under these conditions, although the mass spectra were affected. Then, liquid chromatography-mass spectrometry was used to analyze a PA-oligosaccharide mixture derived from human immunoglobulin G. The PA-oligosaccharides were separated on a reversed-phase column and mass-analyzed directly. The observed molecular weights were close to or identical to those expected from the structures, which were estimated from the elution position on HPLC. This method is rapid and simple, as the mass spectrometer can give the accurate molecular weight of each PA-oligosaccharide in one chromatography run, even if the HPLC separation is incomplete. This method can be used to extend the so-called two-dimensional mapping of PA-oligosaccharides. The structure can be studied in greater detail by tandem MS.     

A reverse-phase high-performance liquid chromatography assay for dihydroxy-acid dehydratase

A sensitive method for assaying dihydroxy-acid dehydratase activity is described. This enzyme produces alpha-ketoisovaleric and alpha-keto-beta-methylvaleric acids, respectively, in the biosynthesis of valine and isoleucine. These alpha-keto acids, after derivatization with 2,4-dinitrophenyl-hydrazine, were separated and quantified by reverse-phase high-performance liquid chromatography on a Zorbax octadecylsilane C-18 column. As little as 50 pmol of alpha-ketoisovaleric was detected in assays using cell-free extracts from Escherichia coli, whose measured specific activity was 8 mumol of alpha-ketoisovaleric acid produced per hour per milligram protein.     

Bacteriocin detection by liquid chromatography/mass spectrometry for rapid identification

Aims: To establish a new system to detect and identify bacteriocins in the early stage of screening for novel bacteriocins. Methods and Results: Liquid chromatography/mass spectrometry (LC/MS) was employed for development of a new system for rapid detection and identification of bacteriocins. The system detected and identified bacteriocins such as nisin and lacticin 481 from 25 μl of culture supernatants of their producing strains by accurate mass determination coupled with simultaneous impurity removal within 40 min. Especially, the system clearly distinguished three nisin variants (A, Z, Q) in culture supernatants of their producing strains, although they have similar structures and molecular masses. Each one‐step pretreatment by cell adsorption–desorption or acetone precipitation improved bacteriocin detection dramatically, especially for mundticin KS. This system could be applied for detection and molecular mass determination of novel bacteriocins by extracting bacteriocin‐related ions. Conclusions: The developed system could detect and identify some kinds of bacteriocin from culture supernatants or pretreated samples. Significance and Impact of the Study: The developed system helps us to identify bacteriocins in the early stage of screening without any or with one‐step pretreatment. This system is effective on not only detection of known bacteriocins but also identification of novel bacteriocins. Consequently, this system will accelerate discovery of novel bacteriocins.