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.     

A sensitive fluorometric assay for dopamine-β-hydroxylase activity by high-performance liquid chromatography

A sensitive and specific fluorometric assay for dopamine-β-hydroxylase (DBH) activity is described. The main natural substrate, dopamine (DA), was used and incubated under optimal conditions. Norepinephrine (NE) formed enzymatically from DA was isolated by an aluminum oxide column and was analyzed by high-performance liquid chromatography (HPLC) with trihydroxyindole fluorescence. Epinephrine (EN) was added to the incubation mixture as an internal standard after incubation, and this assay was therefore highly reproducible. HPLC conditions were settled to elute the product, NE, prior to the substrate, DA, and the internal standard, EN, between NE and DA. Only catecholamines produced significant peaks, and therefore, this assay is highly specific. We applied this method to measure the DBH activity in human serum and cerebrospinal fluid.     

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.     

Automatic computation of enzyme kinetics by HPLC

Enzyme activity can be easily measured by HPLC using tracesof the product itself as an internal standard. Our procedureinvolved the development of an equation using the experimentaldata obtained in the kinetic assay. The entire procedure canthus be automated and a computer program is presented here forfacilitating the assay and saving time. The determination ofthe activity of NAD kinase is reported as an example. Received on March 15, 1990; accepted on May 17, 1990     

Determination of relative rate constants for in vitro RNA processing reactions by internal competition

Studies of RNA recognition and catalysis typically involve measurement of rate constants for reactions of individual RNA sequence variants by fitting changes in substrate or product concentration to exponential or linear functions. A complementary approach is determination of relative rate constants by internal competition, which involves quantifying the time-dependent changes in substrate or product ratios in reactions containing multiple substrates. Here, we review approaches for determining relative rate constants by analysis of both substrate and product ratios and illustrate their application using the in vitro processing of precursor transfer RNA (tRNA) by ribonuclease P as a model system. The presence of inactive substrate populations is a common complicating factor in analysis of reactions involving RNA substrates, and approaches for quantitative correction of observed rate constants for these effects are illustrated. These results, together with recent applications in the literature, indicate that internal competition offers an alternate method for analyzing RNA processing kinetics using standard molecular biology methods that directly quantifies substrate specificity and may be extended to a range of applications.     

Determination of epimedin C in rat plasma by reversed-phase high-performance chromatography after oral administration of Herba Epimedii extract

A simple high-performance liquid chromatographic (HPLC) method has been developed for the determination of epimedin C in rat plasma and applied to a pharmacokinetic study in rats after administration of Herba Epimedii extract. After addition of carbamazepine as an internal standard plasma samples were extracted with ethyl acetate. HPLC analysis of the extracts was performed on a Hypersil ODS2 analytical column using acetonitrile -0.4% acetic acid (25:75, v/v) as the mobile phase. The UV detector was set at 260 nm. The standard curve was linear over the range 0.05-4.0 microg/mL. The lower limit of quantification was 0.05 microg/mL. The HPLC method developed could be easily applied to the determination and pharmacokinetic study of epimedin C in rat plasma after giving the animals Herba Epimedii extract.     

Use of lysozyme as a standard for evaluating the effectiveness of a proteomics process

Automated sequencing of unknowns in bottom-up proteomics makes the data produced susceptible to process control errors, which can be propagated into mistakes in analyte identification. Inclusion of an unintrusive internal standard, such as lysozyme, allows monitoring all phases of the proteomics process including sample preparation, enzymatic digestion, HPLC, mass spectrometry, and database searching. By using this internal standard, digestion issues including rearrangements, semi-tryptic fragments, and modifications were monitored. In addition, control of the HPLC process including column performance was achieved. The use of the lysozyme standard allowed easy optimization of mass spectral conditions including data dependent and collision induced dissociation settings. The use of this internal standard in a study of differential protein expression in rat serum samples is presented.     

The evaluation of inhibitive effectiveness of the tumour necrosis factor-α converting enzyme selective inhibitors by HPLC

A novel high-performance liquid chromatography (HPLC) method based on the internal standard method was established for assaying the tumour necrosis factor-α converting enzyme (TACE) activity and matrix metalloprotease-9 (MMP-9) activity, and was used to evaluate the inhibitive effectiveness of inhibitors to TACE and MMP-9. In the assay method for TACE and MMP-9, peptides labelled with the ultraviolet group-Dpa were used as substrates. Alanine-Dpa was synthesised and was used as the internal standard for quantitative analysis. After the peptide substrates were hydrolysed by TACE (MMP-9) for 15 min (25 min) at 37 °C, the amount of remaining substrates were determined by reversed-phased HPLC with UV detection at 353 nm. The relative peak area of the substrate was linearly dependent on the substrate concentration. This method was then applied to determine the 50% inhibitory concentration (IC??) of GM6001 and inhibitor A for both TACE and MMP-9.     

An Improved Fluorometric High-Performance Liquid Chromatography Method for Sialic Acid Determination: An Internal Standard Method and Its Application to Sialic Acid Analysis of Human Apolipoprotein E

An improved fluorometric HPLC method for sialic acid determination was developed by employing synthetic N-propionylneuraminic acid (NPNA) as an internal standard. A fixed amount of NPNA was added to a sialoglycoconjugate sample. After hydrolyzing sialioglycoconjugates with diluted sulfuric acid, the released sialic acids and NPNA were derivatized with a fluorogenic compound, 1,2-diamino-4,5-(methylenedioxy)benzene (DMB), followed by fluorometric HPLC. The fluorescent derivative of NPNA was separated from those of N-acetylneuraminic acid, N-glycolylneuraminic acid, 2-keto-3-deoxy-D-glycero-D-galacto-nonoic acid, and 2-keto-3-deoxyoctanoate on HPLC. The separation of NPNA derivative on HPLC was not interfered by components of biological samples such as human sera. Using this internal standard method, low amounts of NANA (0.15-1.0 ng) were quantified with the coefficient of variation values below 4%. Using this method, the sialic acid content of human apolipoprotein E was successfully determined. The present method is useful for sensitive and accurate quantification of sialic acids of different molecular species in biological samples.     

Quantitative determination of olanzapine in rat brain tissue by high-performance liquid chromatography with electrochemical detection

A sensitive assay was developed for the measurement of olanzapine in rat brain tissue using HPLC with electrochemical detection. The assay has a lower limit of quantitation of 0.5 ng/ml in tissue homogenate and utilizes a liquid–liquid extraction followed by reversed-phase HPLC for the quantitative analysis of olanzapine. The method provided a linear response for olanzapine over a concentration range of 0.5–100 ng/ml with a coefficient of determination (r²) greater than 0.9995. The extraction efficiencies of olanzapine and internal standard (LY170158) were greater than 82% in brain tissue. The intra-assay and inter-assay relative errors ranged from −5.38 to 17.60% and −3.25 to 10.53%, respectively. The intra-assay and inter-assay RSD values were in the range of 1.12 to 6.96% and 3.78 to 6.68%. Long-term stability studies showed that brain tissue homogenate samples spiked with olanzapine and internal standard are stable at −70°C for at least 110 days. However, a room temperature stability study showed that olanazapine was not stable in brain homogenate if the sample was exposed at 25°C longer than 2 h. This method has been used for the study of the disposition and pharmacokinetics of olanzapine in male Sprague–Dawley rats.     

Fuzzy reasoning system for fault diagnosis of physiological activities in a cultivating process.

Aiming at development of a system which supports cultivating operations, a method to diagnose physiological activities in a cultivating process is presented, and a fuzzy expert system for diagnosing Lactobacillus casei cultivating process is implemented in this paper. This system can calculate specific rates of cell growth, substrate consumption, and product formation with measuring cell mass concentration, substrate concentration, and product concentration by using a turbidity sensor and HPLC. A database is implemented, where standard curves on specific rates representing characteristics of microorganisms are stored according to normalized substrate consumption. Comparing the calculated specific rates with standard values derived from the database, the system diagnoses physiological activities of the microorganisms. As a case study, a knowledge base for diagnosing lactic acid production process is implemented. The use of fault diagnosis on pH malfunctions by the expert system proves its reasonable performance.     

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.     

A sensitive high-performance liquid chromatographic procedure with fluorometric detection for the analysis of decarboxylated S-adenosylmethionine and analogs in urine samples

A highly sensitive HPLC method for the determination of decarboxylated S-adenosylmethionine (dc-SAM) by fluorometric detection was developed. The reaction of dc-SAM and its analogs with chloroacetaldehyde leads to the corresponding 1,N6-etheno derivatives. These highly fluorescent derivatives were fully characterized through their proton nuclear magnetic resonance spectra and/or mass spectra. This derivatization procedure has been applied to the analysis of dc-SAM in rat and human urine. After a simple cation exchange column prepurification, the urine extracts were derivatized with chloroacetaldehyde and analyzed by reversed-phase HPLC with fluorometric detection. The method allowed the determination of subpicomole amounts of dc-SAM and was shown to be highly reproducible with the use of decarboxylated S-adenosylethionine as internal standard. The application of the method to the analysis of urine of rats treated with MDL 72175, a potent ornithine decarboxylase inhibitor, showed that the dc-SAM levels increased in a dose-related fashion.     

HPLC法测定藜豆中左旋多巴的含量

采用HPLC法建立测定藜豆中左旋多巴含量的方法。色谱柱为岛津C-18甲基硅烷柱(250 min×4.6mm,5μm),流动相为甲醇:0.1 mol／L醋酸溶液(25:75,v／v),以硫唑嘌呤为内标物,检测波长280 nm,流速1.0 mL／min,柱温为室温,供试品的前处理方法为微波辅助提取法。结果表明,该方法准确度高,线性关系好,重现性好,平均加样回收率为97.34％,RSD 1.01％(n=5),结果满意。     

Determination of tianeptine in human plasma using high-performance liquid chromatography with fluorescence detection

A new, selective and sensitive high-performance liquid chromatography (HPLC) method with fluorimetric detection was developed for the determination of tianeptine (TIA) in human plasma using solid phase extraction (SPE) procedures. The method is based on the derivatization of TIA with 4-chloro-7-nitrobenzofurazan (NBD-Cl) in borate buffer of pH 8.5 to yield a yellow, fluorescent product. The HPLC separation was achieved on a Phenomenex C(18) column (250 mm x 4.6 mm) using a mobile phase of acetonitrile-10mM orthophosphoric acid (pH 2.5) (77:23, v/v) solvent system at 1 mL/min flow rate. Gabapentin (GA) was used as the internal standard. The fluorometric detector was operated at 458 nm (excitation) and 520 nm (emission). The assay was linear over the concentration range of 5-300 ng/mL. The detection limit (LOD) was found to be 2 ng/mL. The mean recovery was determined to be 88.6%. The proposed method was applied for pharmacokinetic study of 12.5mg TIA in a healthy volunteer.     

Rapid method for the analysis of itraconazole and hydroxyitraconazole in serum by high-performance liquid chromatography

An assay for the determination of itraconazole and its active metabolite hydroxyitraconazole in serum has been developed, using ketoconazole as internal standard. The procedure involved a one-step liquid-liquid extraction of the drug, its metabolite and the internal standard, followed by their separation by reversed-phase HPLC. In this paper the validation of this method is described.     

Assay of nicotinamide deamidase activity using high-performance liquid chromatography

A rapid, simple and reproducible method has been developed for the determination of nicotinamide deamidase activity using high-performance liquid chromatography (HPLC). Nicotinic acid (NA) liberated from nicotinamide (NAA) after a 15-min enzyme reaction was determined directly by HPLC without further separation steps. Both NA, the product, and NAA, the substrate were separated by reversed-phase ion-pair isocratic chromatography and detected at 261 nm. The present method could be applied to the measurement of deamidase activity in crude cell extracts prepared from several bacterial strains. The Michaelis constant of nicotinamide deamidase in Enterobacter agglomerans was 36 μM for NAA. This method is useful for the measurement of nicotinamide deamidase from various sources.     

Simplified methodology to determine breast milk retinol concentrations

The impact of a nutritional intervention trial on vitamin A status can be evaluated by measuring the total vitamin A concentration in breast milk both before and after the intervention. Because breast milk contains a spectrum of retinyl esters as well as retinol and high lipid content, determination of total breast milk retinol routinely requires saponification with alcoholic potassium hydroxide. Retinol is then extracted with an organic solvent, usually hexanes, before HPLC analysis. Retinyl acetate, although commonly used as an internal standard, is not ideal because it can only be added after saponification and extraction and consequently, will only account for part of the total losses. A method has now been developed that uses 3,4-didehydroretinyl acetate (DRA) as an internal standard. DRA is an excellent choice as an internal standard for the following reasons: 1) DRA can be added to the breast milk before saponification and can be carried through the analysis as dehydroretinol (DR), 2) the percent recovery can be easily determined, and 3) DR is easily separated from retinol during HPLC analysis. The procedure, as described, typically gives a mean extraction efficiency of 80-90%. Moreover, the average coefficient of variation is <5% on the same sample run several times in the same day.