Determination of angiotensin converting enzyme inhibitory activity by high-performance liquid chromatography/electrospray-mass spectrometry

A sensitive and rapid method for determination of angiotensin converting enzyme (ACE) inhibitory activity was developed based on a combination of enzymatic reaction followed by high performance liquid chromatography/electrospray-mass spectrometry (HPLC-ESI-MS) determination of its product. The most commonly used substrate hippuryl-histidyl-leucine (HHL) or hippuryl-glycyl-glycine (HGG) hydrolysis catalyzed by purified rabbit lung ACE or human plasma ACE was investigated in the presence of benazeprilat. The incubation time was 8 min for purified lung ACE, and 16 min for human plasma ACE. The produced hippuric acid (HA) was separated from substrate HHL or HGG by HPLC on a C(18) column with isocratic elution within 6.5 min, and quantified by electrospray ionization mass spectrometry (ESI-MS) with p-phthalic acid as an internal standard (IS). The limit of detection of HA was 6.0 ng/ml. HHL or HGG hydrolysis catalyzed by purified lung ACE displayed excellent accuracy and reproducibility. The small total reaction volume, the low concentration of substrate, and the simple treating procedures present the advantages of the new method. Furthermore, the total time of the whole procedure for one sample with the novel method is less than 1/2 of that of the conventional HPLC or spectrophotometry method, while the accuracy and the precision of the new method are almost the same as the conventional HPLC method with UV detection.     

High-performance liquid chromatography determination of enzyme activities in the presence of small amounts of product

Enzymes can be assayed by HPLC by calculating the amount of substrate(s) left over, or product formed, through the peak area ratios with a suitable internal standard. However, sometimes the substrates used are contaminated with small amounts of products and this can lead to errors in the determination of the enzyme activity. A method for a HPLC test of such enzymes, which prevents eventual errors, uses the ratio substrate/product at time zero as internal standard and the kinetics can be followed with the aid of a simple mathematical equation. This approach was applied to the determination of the activities of papain, urokinase, NAD glycohydrolase, and pyruvate kinase samples and it was compared with the data obtained by the internal standard method, giving reproducible results in all cases.     

Separation of phytanic and pristanic acid by high-pressure liquid chromatography: application of the method

The synthesis of pristanic acid from phytanic acid, and a simple reversed-phase high-pressure liquid chromatographic (HPLC) method for the separation and purification of these acids, is described. A base-line separation of [U-3H]phytanic and [U-3H]pristanic acid is achieved with a graphitized carbon column. The isoprenoid metabolites formed after incubation of cultured fibroblasts with phytanic or pristanic acids are extracted with a Sep-Pak C18 cartridge and separated from the substrates by the same reversed-phase HPLC used for substrate purification. The methods are suitable for studies on the mechanisms for degradation of phytanic acid. Recently, different inborn errors with accumulation of phytanic acid have been defined. The present method will be a useful tool in our efforts to define these metabolic defects and their subcellular localization.     

Quantitation of cytochrome c release from rat liver mitochondria

The apoptogenic protein cytochrome c can be quantitated by reverse-phase HPLC, but this method is not utilized by those who investigate mechanisms of cell death. Here, we extend the sensitivity of the method to exceed that available from immunogenic approaches and report specific procedures for applying the method to preparations of intact mitochondria, and to supernatants and pellets that arise from mitochondrial incubations. The detection limit corresponds to 0.6% of total cytochrome c found in 100 microg of rat liver mitochondrial protein, or to all of the cytochrome c that is expected in approximately 6000 hepatocytes. A single determination can be completed in 20 min, compared to a time scale of days for Western blotting methods, or hours for ELISA-based methods. The procedures are illustrated by experiments that determine the amount of cytochrome c released following the mitochondrial permeability transition as a function of medium ionic strength, and by long-term incubations of intact mitochondria in the presence and absence of an exogenous oxidizable substrate. Swelling and the release of adenylate kinase activity have been determined simultaneously to show how the data can be applied to evaluate the role of outer membrane disruption in mechanisms that release cytochrome c.     

Enzymatic assays for NAD-dependent deacetylase activities

The NAD-dependent deacetylases are a new class of enzymes responsible for the removal of acetyl groups from lysines on proteins. Instead of water, the NAD-dependent deacetylases use a highly reactive ADP-ribose intermediate as a recipient for the acetyl group. The products of the reaction are nicotinamide, acetyl-ADP-ribose, and a deacetylated substrate. Many assays have been developed for the measurement of NAD-dependent deacetylase activity. In this review we present assays based on each of the two reactions catalyzed by these enzymes, deacetylation and NAD hydrolysis. First we describe methods for the production of acetylated protein and peptide substrates for use in deacetylation reactions. Then we describe four methods for assaying deacetylation, three of which directly measure the loss of acetyl groups from a protein or peptide substrate, and one that measures acetate production. We also describe two indirect methods for following enzyme activity, NAD hydrolysis and a novel NAD-nicotinamide exchange reaction. Finally, a quantitative method using a monoacetylated peptide as a substrate and HPLC to measure products is described.     

New and highly sensitive assay for l-5-hydroxytryptophan decarboxylase activity by high-performance liquid chromatography—voltammetry

This paper describes a new, inexpensive and highly sensitive assay for aromatic l-amino acid decarboxylase (AADC) activity, using l-5-hydroxytryptophan (l-5-HTP) as substrate, in rat and human brains and serum by high-performance liquid chromatography (HPLC) with voltammetric detection. l-5-HTP was used as substrate and d-5-HTP for the blank. After isolating serotonin (5-HT) formed enzymatically from l-5-HTP on a small Amberlite CG-50 column, the 5-HT was eluted with hydrochloric acid and assayed by HPLC with a voltammetric detector. N-Methyldopamine was added to each incubation mixture as an internal standard. This method is sensitive enough to measure 5-HT, formed by the enzyme, 100 fmol to 140 pmol or more. An advantage of this method is that one can incubate the enzyme for longer time (up to 150 min), as compared with AADC assay using l-DOPA as substrate, resulting in a very high sensitivity. By using this new method, AADC activity was discovered in rat serum.     

New and highly sensitive assay for l-5-hydroxytryptophan decarboxylase activity by high-performance liquid chromatography—voltammetry

This paper describes a new, inexpensive and highly sensitive assay for aromatic l-amino acid decarboxylase (AADC) activity, using l-5-hydroxytryptophan (l-5-HTP) as substrate, in rat and human brains and serum by high-performance liquid chromatography (HPLC) with voltammetric detection. l-5-HTP was used as substrate and d-5-HTP for the blank. After isolating serotonin (5-HT) formed enzymatically from l-5-HTP on a small Amberlite CG-50 column, the 5-HT was eluted with hydrochloric acid and assayed by HPLC with a voltammetric detector. N-Methyldopamine was added to each incubation mixture as an internal standard. This method is sensitive enough to measure 5-HT, formed by the enzyme, 100 fmol to 140 pmol or more. An advantage of this method is that one can incubate the enzyme for longer time (up to 150 min), as compared with AADC assay using l-DOPA as substrate, resulting in a very high sensitivity. By using this new method, AADC activity was discovered in rat serum.     

Development of a microtiter plate-based method for determination of degradation profile of nitrophenolic compounds

A microtiter plate-based assay was developed for the automatic monitoring of degradation profile of the yellow-coloured nitrophenolic compounds. The method enables to reduce the intervals between measurements of substrate concentration to minutes and to overcome the problem of discontinuity of sampling typical for conventional methods. The concentrations of nitrophenolic compounds were calculated from the absorbance values determined automatically by BIOSCREEN C. Verification of the method was based on the comparison of results with the conventional HPLC method results. The values of the rate and saturation constants were comparable for both the microtiter plate-based assay and the conventional HPLC method. The automatic method described here seems to be efficient for the screening degradation studies, which requires the treatment of quantity of samples.     

Simple and sensitive high-performance liquid chromatography method for the determination of docetaxel in human plasma or urine

Several methods for quantification of docetaxel have been described mainly using HPLC. We have developed a new isocratic HPLC method that is as sensitive and simpler than previous methods, and applicable to use in clinical pharmacokinetic analysis. Plasma samples are spiked with paclitaxel as internal standard and extracted manually on activated cyanopropyl end-capped solid-phase extraction columns followed by isocratic reversed-phase HPLC and UV detection at 227 nm. Using this system, the retention times for docetaxel and paclitaxel are 8.5 min and 10.5 min, respectively, with good resolution and without any interference from endogenous plasma constituents or docetaxel metabolites at these retention times. The total run time needed is only 13 min. The lower limit of quantification is 5 ng/ml using 1 ml of plasma. The validated quantitation range of the method is 5–1000 ng/ml with RSDs≤10%, but plasma concentrations up to 5000 ng/ml can be accurately measured using smaller aliquots. This method is also suitable for the determination of docetaxel in urine samples under the same conditions. The method has been used to assess the pharmacokinetics of docetaxel during a phase I/II study of docetaxel in combination with epirubicin and cyclophosphamide in patients with advanced cancer.     

7,7,8,8-Tetracyanoquinodimethane as a new derivatization reagent for high-performance liquid chromatography and thin-layer chromatography: rapid screening of plasma for some antidepressants

Using 7,7,8,8-tetracyanoquinodimethane (TCNQ) as a new derivatization reagent for HPLC and TLC, novel methods are described to detect secondary amine-bearing antidepressants (paroxetine, desipramine, fluoxetine, nortriptyline, maprotiline). The HPLC method is sensitive enough to detect these drugs in plasma at therapeutic levels whereas the latter has potential to detect them in overdose or forensic cases. The methods are based on purple chromogens formed by the displacement reaction of the drugs with TCNQ. The resulting chromogens are directly separated by either reversed-phase HPLC on a C(18) column or TLC on silicagel plates. For HPLC, acetonitrile-water (60:40) was used as mobile phase, with detection at 567 nm and separation in 40 min. For TLC, three developing solvent systems were used. By HPLC, 36 ng ml(-1) spiked plasma concentration of the drugs gave easily detectable signals whereas by TLC, detection limits varied mostly between 240 and 480 ng ml(-1). The HPLC method was applied to real plasma samples. The methods described are simple and very selective; some metabolites of these antidepressants and a vast number of drugs do not interfere with detection.     

High-performance liquid chromatographic method for measurement of cytochrome P450-mediated metabolism of 7-ethoxy-4-trifluoromethylcoumarin

An HPLC method for analysis of deethylation of 7-ethoxy-4-trifluoromethylcoumarin (ETFMC), a substrate of various enzymes of the cytochrome P450 superfamily, was developed. ETFMC was incubated at 37°C with human hepatic microsomes or microsomes prepared from a lymphoblastoid cell line that expresses human CYP2B6. Under these conditions, the highly fluorescent metabolite 7-hydroxy-4-trifluoromethylcoumarin (HTFMC) is formed. The metabolite was analyzed by reversed-phase HPLC with fluorescence detection. The limits of detection of the metabolite were 5.0 fmol per injection, a sensitivity at least one order of magnitude greater than the standard method, which does not involve HPLC. This method will be of great utility when quantities of microsomal protein from cell lines expressing human CYP enzymes are limited.     

Internal-surface reversed-phase chromatography for plasma metabolite analysis of radiopharmaceuticals

The use of internal-surface reversed-phase (ISRP) chromatography of unprocessed plasma samples was investigated as an alternative method of quantitation of the arterial plasma metabolite time course of [^18F]N-methylspiperone. The ISRP method was directly compared to standard solid phase extraction/HPLC (SPE/HPLC) methods currently in wide use. Results indicate that: (1) the ISRP method is rapid and minimizes sample preparation; (2) recovery of radioactivity from the ISRP column is > 90%; (3) no radioactivity remains associated with chromatographically size excluded proteins and (4) the quantitative results are well correlated with conventional SPE/HPLC methods.     

Direct enantioselective HPLC monitoring of lipase-catalyzed kinetic resolution of tiaprofenic acid in nonstandard HPLC organic solvents

The first straightforward lipase-catalyzed enantioselective access to enantiomerically enriched tiaprofenic acid as a versatile method in chiral separation of racemates is demonstrated. The latter was directly monitored by enantioselective HPLC using a 3,5-dimethylphenylcarbamate derivative of cellulose-based chiral stationary phase namely Chiralpak IB (the immobilized version of Chiralcel OD). Non-standard HPLC organic solvents were used as diluent to dissolve the "difficult to dissolve" enzyme substrate (the acid) and as eluent for the simultaneous enantioselective HPLC baseline separation of both substrate and product in one run without any further derivatization. The existence of a non-standard HPLC organic solvent (e.g., methyl tert-butyl ether) in the mobile phase composition is mandatory to accomplish the simultaneous enantioselective HPLC baseline separation of both substrate and product.     

Quantitative HPLC analysis of acidic opines by phenylthiocarbamyl derivatization

A new method for the quantitative analysis of acidic opines--alanopine, strombine, tauropine, and beta-alanopine--is presented. The method is based on formation of phenylthiocarbamyl (PTC) derivatives of the acidic opines after partial purification by ion-exchange chromatography. The PTC acidic opines are separated by reverse-phase high-performance liquid chromatography (HPLC) and detected within 20 min by ultraviolet absorbance. This HPLC method gives higher sensitivity, 10-30 pmol minimum detection, and better reproducibility than the high-voltage paper electrophoresis method. There is also good agreement for the three acidic opines (alanopine, strombine, and tauropine) when compared by HPLC and electrophoresis methods. Accumulation of beta-alanopine was observed for the first time in the adductor muscle of blood shell, Scapharca broughtonii, during aerial exposure by application of the HPLC detection method.     

Determination of amphetamine in human urine by dansyl derivatization and high-performance liquid chromatography with fluorescence detection

A simple procedure for the determination of amphetamine in urine with minimal sample preparation is described. This method involves direct addition of human urine to an acetone-dansyl chloride solution for simultaneous deproteinization and fluorescence derivatization. The derivatized amphetamine is then measured by HPLC with fluorescence detection. It eliminates the extraction procedures often required by other HPLC or GC methods. The effects of pH, temperature and reaction time on the derivatization reaction were investigated. The stability of amphetamine-dansyl chloride in different storage conditions was examined. The detection limit and linearity associated with this assay are discussed.     

Measurement of 25-hydroxyvitamin D in the clinical laboratory: Current procedures, performance characteristics and limitations

In this review we describe procedures, performance characteristics and limitations of methods available for the measurement of 25-hydroxyvitamin (25OHD) since the year 2000. The two main types of methods are competitive immunoassay and those based on chromatographic separation followed by non-immunological direct detection (HPLC, LC-MS/MS). Lack of a reference standard for 25OHD has, until recently, been a major issue resulting in poor between-method comparability. Fortunately this should soon improve due to the recent introduction of a standard reference material in human serum (SRM 972) from the National Institute of Standards and Technology (NIST). For immunoassay, specificity can be an issue especially in relation to the proportion of 25OHD2 that is quantified whereas HPLC and LC-MS/MS methods are able to measure the two major vitamin D metabolites 25OHD2 and 25OHD3 independently. HPLC and LC-MS/MS require more expensive equipment and expert staff but this can be offset against lower reagent costs. Increasingly procedures are being developed to semi-automate or automate HPLC and LC-MS/MS but run times remain considerably longer than for immunoassays especially if performed on automated platforms. For most HPLC and LC-MS/MS methods extraction and procedural losses are corrected for by the inclusion of an internal standard which, in part, may account for higher results compared to immunoassay. In general precision of immunoassay, HPLC and LC-MS/MS are comparable and all have the required sensitivity to identify severe vitamin D deficiency. Looking to the future it is hoped that the imminent introduction of a standard reference method (or methods) for 25OHD will further accelerate improvements in between method comparability.     

Direct enantioselective HPLC monitoring of lipase‐catalyzed kinetic resolution of flurbiprofen

The solvent versatility of Chiralpak IB, a 3,5‐dimethylphenylcarbamate derivative of cellulose‐based chiral stationary phase, is demonstrated in the direct enantioselective HPLC monitoring of lipase‐catalyzed kinetic resolution of flurbiprofen in nonstandard HPLC organic solvents. Nonstandard HPLC organic solvents were used as the reaction media for the lipase‐catalysis and in mean time as diluent to dissolve the “difficult to dissolve” enzyme substrate (the acid) and as eluent for the simultaneous enantioselective HPLC baseline separation of both substrate and product in one run without any further derivatization. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Merits of HPLC-based method over spectrophotometric method for assessing the kinetics and inhibition of mammalian adenosine deaminase

Measurement of adenosine deaminase (ADA) activity using spectrophotometric method presents problem, regarding the quantitative estimation of the substrate degradation and product formation, due to the closely apposed lambda(max) of the substrates, product and the inhibitor. The feasibility of applying reverse-phase HPLC technique, for studying adenosine deaminase-catalyzed reaction product and inhibition study was examined. We have drawn a comparison between the HPLC-based method over the corresponding spectrophotometric method. A gradient elution pattern was used to separate substrate (adenosine and deoxyadenosine), product (inosine and deoxyinosine) and standard adenosine deaminase inhibitor (erythro-9-(3-nonyl-p-aminobenzyl)-adenine) in the HPLC method. The product formation was quantitated by monitoring the absorbance at 260 nm with the progress of time. The limit of detection as well as the limit of quantification of the respective enzymatic product were found to be in nano molar (nM) range in the HPLC method. This study was also extended to monitor adenosine deaminase activity in different cancer cells of hematological origin. The HPLC-based method is found to be suitable for the quantitative estimation of adenosine deaminase-catalyzed reaction product and for studying inhibition mechanism of different inhibitors. The HPLC-based method has specific advantages over the spectrophotometric method. Moreover, the concentration of different nucleotides in cell lysate and body fluid can be measured using this HPLC method.     

High-performance liquid chromatography separation of phospholipid classes and arachidonic acid on cyanopropyl columns

An HPLC method for the separation and analysis of arachidonic acid and eight phospholipid classes is described: phosphatidylglycerol, phosphatidylinositol, cardiolipin, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, and 2-lysophosphatidylcholine. The separation is carried out at 60 degrees C on 2 cyanopropyl columns using a gradient of acetonitrile and 5 mM sodium acetate (pH 5.0). Cyanopropyl columns require a lower proportion of water in the mobile phase to elute the more polar phospholipids than other types of columns and are thus less prone to equilibration problems. The method is highly reproducible (average coefficient of variation for each retention time less than or equal to 3.5%) and permits analysis of peaks by phosphorus content. Data obtained by analyzing lipid extracts from rat alveolar macrophages prelabeled with [G-3H]-arachidonic acid were analyzed by this HPLC method and compared to standard analysis by TLC. There was a significant correlation between the radioactivity profiles obtained with the two chromatographic methods (HPLC versus TLC) by linear regression analysis [HPLC = 0.83 (TLC) + 3.58, n = 25, r = 0.95, P less than 0.001].     

High-performance liquid chromatographic method to measure protein L-isoaspartyl/D-aspartyl o-methyltransferase activity in cell lysates

Protein l-isoaspartyl/d-aspartyl o-methyltransferase (PIMT) is a widely expressed protein repair enzyme that restores isomerized aspartyl residues to their normal configuration. Current methods for measuring PIMT activity have limited sensitivity or require radioactivity. We have developed a highly sensitive new assay method to measure PIMT activity in cell lysates. As a substrate, we used a fluorescently labeled delta sleep-inducing peptide (DSIP) that contains an isoaspartyl residue: 7-nitro-2,1,3-benzoxadiazole (NBD)-DSIP(isoAsp). The PIMT-catalyzed transfer of a methyl group onto this substrate can be detected with a simple high-performance liquid chromatography (HPLC) procedure. After the enzyme reaction, the methylated form of the peptide is stable and can be reproducibly separated from the unmethylated form in an acidic solvent and fluorometrically detected by HPLC. The limit of detection was estimated to be approximately 1 pmol of NBD-DSIP(isoAsp) (signal/noise ratio [S/N] = 3), and the quantitation limit of the activity was approximately 18 μg of total cell lysate from HEK293 cells (10.7 pmol/min/mg protein). This assay method is sensitive enough to detect PIMT activity in biological samples without the use of radioisotopes, offering significant advantages over previously reported methods.