Present HPLC Method Research Articles

Chiral-phase HPLC separation of (divinyl-)protochlorophyllide-a enantiomers as key precursors in chlorophyll biosynthesis from their 132-stereoisomeric prime forms

Protochlorophyllide(PChlide)-a and its 8-vinylated analog, divinyl(DV)-PChlide-a, are common and essential intermediates in the biosynthesis of all naturally occurring chlorophyll (Chl) pigments. These porphyrinoid-type pigments have a single optically active (asymmetric) carbon atom at the 132-position, so their stereoisomers are (132R)- and (132S)-enantiomers. The former and latter are called (DV-)PChlide-a and (DV-)PChlide-a′, respectively. In this study, chiral-phase HPLC separation of enantiomeric (DV-)PChlides-a/a′ was demonstrated. The (132R)-enantiomeric PChlide-a was eluted more slowly than the corresponding (132S)-enantiomeric PChlide-a′ under the present HPLC conditions. On the other hand, the elution order of (132R)-DV-PChlide-a and (132S)-DV-PChlide-a′ was reverse to that of PChlides-a/a′. After the separation of each enantiomer by the chiral-phase HPLC, the stereoisomeric configuration at the 132-position was characterized by means of circular dichroism spectroscopy. The present chiral-phase HPLC method enables us to evaluate optical purities of (DV-)PChlide-a species. For example, PChlide-a and/or DV-PChlide-a extracted from the spent medium and harvested cells of cultured purple photosynthetic bacterial mutants, the former of which has been often used as the source of (DV-)PChlide-a substrates for enzymatic reactions, were revealed to be mostly racemized, giving enantiomeric mixtures of (DV-)PChlides-a/a′.

Determination of Hydrochlorothiazide and Two Major Degradation Products by Stability Indicating High Performance Liquid Chromatography

Background: Hydrochlorothiazide (HCTZ) is a thiazide diuretic which comprises two sulfonamide groups. The literature is not clear regarding the identification of the chromatographic peaks of its two major related substances: chlorothiazide and 4-amino-6-chloro-1,3-benzenedisulfonamide (DSA). Methods: In the present study, a simple, sensitive, and selective HPLC method was developed and validated for the assay of HCTZ, Chlorothiazide and DSA. The method was carried out on a C18 column, maintained at 40ºC. The mobile phase was composed of monobasic potassium phosphate buffer 0.02M pH 3.0/acetonitrile/methanol (82:9:9, v/v/v), run at a flow rate of 1.0 mL/min, and UV detection at 270 nm. Results: All related compounds including processing impurities and degradants from stressed samples were well separated from each other. The performance of this method was validated in accordance to the ICH guidelines and included specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness. Conclusion: Based on the results, the HCTZ degradation pathway was proposed and the validated HPLC method was successfully applied to the quantitative analysis of HCTZ in pharmaceutical formulations, contributing to improve quality control, to assure therapeutic efficacy and to clarify the literature.

Development and validation of chiral HPLC method for quantitative determination of hydrazine analogue in Argatroban monohydrate

Abstract Argatroban is an anticoagulant which is used as direct thrombin inhibitor. It consists of hydrazine analogue, which is probably similar to Argatroban. The single HPLC method is already reported in to United States Pharmacopeia (USP). In that method, Argatroban peak eluted as R-Argatroban and S-Argatroban while hydrazine analogue eluted as P1 and P2 peaks. The P2 peak of Hydrazine analogue is eluted as retention time of R-Argatroban peak. Therefore it was difficult to determine the accurate concentration of Hydrazine analogue in to Argatroban. The existing method is longer run time up to 102 min. The present HPLC method shows appropriate resolution between hydrazine analogue and Argatroban. The total run time reduces up to 40 min, which is a ∼2.5 time lesser than reported in literature. In present method hydrazine analogue and Argatroban are eluted to be 9.0 min and 12.5 min respectively. The effective chromatographic separations are achieved on YMC chiralart cellulose-SC, 250×4.6 mm, 5μ column and normal phase with linear elution. The mobile phase was uniform mixture of methanol, acetonitrile, dichloromethane, trifluoroacetic acid, triethylamine with ratio of 30:50:920:3:4 and delivered flow rate 0.7 mL/min. Analysis is performed on UV/PDA detector with detection wavelength is adjusted at 259 nm. The injection volume is 5μL and column oven temperature is 35°C. The present method is accurate and precise as well as linear in the range of LOQ to 150 % level. The finalized concentration of hydrazine analogue for limit of detection and quantitation is 0.003 and 0.010%. The correlation coefficient of hydrazine analogue is 0.99947. The recovery for ranged between 100.00 to 103.29%. In robustness study for of mobile phase flow rate and column oven temperature, no significant changes are observed in system suitability parameters and result. This method has been developed and validated using the ICH guideline Q2(R1).The new method is sensitive, precise, accurate, and rapid; it also qualifies all the criteria of linearity, stability, as well as robustness.

Development of validated RP HPLC method with fluorescence detection for simultaneous quantification of sacubitril and valsartan from rat plasma

ABSTRACTIn the present work, a sensitive, accurate and precise RP HPLC method has been established for simultaneous determination of sacubitril and valsartan from rat plasma by using irbesartan as an internal standard. Separation of analytes were carried out on monolithic column using 10 mM phosphate buffer (pH 2.6), methanol and acetonitrile in a proportion of 50:25:25 (v/v). Analytes were monitored using fluorescence detector maintained at an excitation wavelength of 249 nm and an emission wavelength was set at 380 nm till the elution of valsartan as well as internal standard and switched online to 320 nm for sacubitril. Analysis of analytes from rat plasma was carried out by protein precipitation using methanol and acetonitrile. Valsartan and sacubitril showed good linearity in the concentration range of 1–250 ng/ml and 1–200 ng/ml respectively, with good correlation coefficient (r2 ≥ 0.998). Further, the precision and accuracy of the proposed procedure were suitable as the percent relative standard deviation and percent relative error were well within the acceptable range. The average percent of recovery from the rat plasma was found to be 96.6% and 97.6% for valsartan and sacubitril respectively. The newly proposed method can be used for regular pharmacokinetic studies because of simplicity in sample preparation, short analysis time (<5 min) and good sensitivity.

Simultaneous determination of serum propafenone and its metabolites using high-performance liquid chromatography.

Propafenone, a class Ic antiarrhythmic agent, is metabolized to 5-hydroxypropafeone (5-OHP) and N-depropylpropafenone (NDPP). Simultaneous determination of serum propafenone and its metabolites was performed using HPLC equipped with a conventional octadecylsilyl silica column and ultraviolet detector. The wavelength was set at 250 nm. Propafenone and its metabolites in the serum were extracted using diethyl ether. The mobile phase solution, comprising 1-pentanesulfonic acid sodium salt (0.1m), acetonitrile and acetic acid (280:185:2.5, v/v/v), was pumped at a flow rate of 1 mL/min. The recoveries of propafenone, 5-OHP and NDPP were greater than 85, 82 and 60%, respectively, with the coefficients of variation (CVs) less than 5.4, 1.9 and 2.9%, respectively. The calibration curves were linear for a concentration range of 12.5-1500 ng/mL for propafenone and 2-500 ng/mL for 5-OHP and NDPP (r > 0.999). CVs in the intraday assays were 1.0-3.8% for propafenone, 0.6-2.0% for 5-OHP and 0.6-1.7% for NDPP. CVs in interday assays were 1.3-7.7% for propafenone, 1.1-6.5% for 5-OHP and 5.4-8.0% for NDPP. The present HPLC method can be used to assess the disposition of propafenone and its metabolites for pharmacokinetic studies and therapeutic drug monitoring of propafenone.

Determination of Coenzyme A and Acetyl-Coenzyme A in Biological Samples Using HPLC with UV Detection.

Coenzyme A (CoA) and acetyl-coenzyme A (acetyl-CoA) play essential roles in cell energy metabolism. Dysregulation of the biosynthesis and functioning of both compounds may contribute to various pathological conditions. We describe here a simple and sensitive HPLC-UV based method for simultaneous determination of CoA and acetyl-CoA in a variety of biological samples, including cells in culture, mouse cortex, and rat plasma, liver, kidney, and brain tissues. The limits of detection for CoA and acetyl-CoA are >10-fold lower than those obtained by previously described HPLC procedures, with coefficients of variation <1% for standard solutions, and 1–3% for deproteinized biological samples. Recovery is 95–97% for liver extracts spiked with Co-A and acetyl-CoA. Many factors may influence the tissue concentrations of CoA and acetyl-CoA (e.g., age, fed, or fasted state). Nevertheless, the values obtained by the present HPLC method for the concentration of CoA and acetyl-CoA in selected rodent tissues are in reasonable agreement with literature values. The concentrations of CoA and acetyl-CoA were found to be very low in rat plasma, but easily measurable by the present HPLC method. The method should be useful for studying cellular energy metabolism under normal and pathological conditions, and during targeted drug therapy treatment.

DEVELOPMENT AND VALIDATION OF CARBAMAZEPINE PLASMA CONCENTRATIONS MEASUREMENT AND ITS APPLICATION ON EPILEPSY PATIENTS

Objective: To develop and validate high-performance liquid chromatography with photodiode array (HPLC-PDA) detector as a method for measuring carbamazepine plasma concentrations in epilepsy patients treated with monotherapy or polytherapy.Methods: Carbamazepine was extracted from epilepsy patients’ plasma through liquid-liquid extraction, using protein precipitation with chloroform. Analysis was performed using HPLC with Inertsil DS-4 C18 (4.6x150 mm), 5 μm particle size column. The optimal condition for separation was established in a mobile phase consisting of acetonitrile: water (50:50) at a flow rate of 1.0 ml/min, detected by PDA detector at 220 nm. Propylparaben was used as the internal standard. The retention time was 3.5 min.Results: Linearity was obtained over a concentration range of 0.5-16 μg/ml with r = 0.999. The method showed good intra-and inter-day precision and accuracy of more than 90% difference (% diff) and 95% relative standard deviation (RSD). Lower limit of quantification (LOQ) was 0.5 μg/ml and lower limit of detection (LOD) was 0.2 μg/ml with 100% accuracy and more than 90% precision. Recovery test was nearly 100%. Stability of carbamazepine plasma concentration in 3 epilepsy patients was measured on the first and third month of treatment, ranging between 83.5 to 98.7%. When used to compare carbamazepine as a monotherapy versus polytherapy, the method showed good selectivity.Conclusion: The present HPLC method was valid for measuring carbamazepine plasma concentrations in epilepsy patients treated with monotherapy or polytherapy. This method meets the standard in the EMEA guideline in terms of linearity, precision, and accuracy, also selectivity in epilepsy patients treated with polytherapy.

Rapid determination of the angiotensin I-converting enzyme inhibitory activity of peptide by HPLC method: A simulated gastrointestinal digestion study

ABSTRACTAngiotensin I-converting enzyme (ACE) plays a vital role in blood pressure regulation. In vitro ACE inhibitory activity assays are commonly used for evaluating the activity of inhibitors in many researches. In the present study, a rapid, sensitive, simple, and accurate HPLC method for ACE inhibitory activity determination using chicken meat as a model was developed. Specific attention was paid to assess the ACE inhibitory activity of peptides released during simulated gastrointestinal digestion. Using hippuryl–histidyl–leucine as the substrate, ACE can catalyze it into hippuric acid, which is rapidly and sensitively detected by the HPLC at the retention time of 8–10 min. The ACE inhibitory activity of each digestion solution can be rapidly determined every 15 min or less. The model protein displayed a high ACE inhibitory activity during simulated gastrointestinal digestion, but the digestion solution in the stomach showed stronger activity than in the intestine.

Simultaneous quantification and validation of new peroxynitrite scavengers from Artemisia iwayomogi

Context: Artemisia iwayomogi Kitamura (Compositae) has been very widely used for the treatment of acute or chronic hepatitis, jaundice, and gastritis. In the course of our continuing efforts to identify and quantify peroxynitrite scavengers from Compositae plants, A. iwayomogi was used in this study.Objective: The present study was aimed to identify and quantify the peroxynitrite scavengers of A. iwayomogi.Materials and methods: Silica gel and ODS were used for column chromatography. The isolated compounds were quantified using an HPLC equipped with a Capcell Pak C18 column (5 μm, 250 mm × 4.6 mm i.d.), and the method was validated for the quality control. Peroxynitrite (ONOO−)-scavenging activities of the compounds and extracts were evaluated on the measurement of highly fluorescent rhodamine 123 converted from non-fluorescent dihydrorhodamine (DHR)-123 under the presence of peroxynitrite.Results: Based on the spectroscopic evidences, a new compound, 2″-O-caffeoylrutin (2″-O-trans-caffeic acid ester of quercetin 3-O-α-l-rhamnopyranosyl(1 → 6)-β-d-glucopyranoside) was isolated and determined together with patuletin 3-O-glucoside, scopolin, scopoletin, rutin, 3,4-dicaffeoylquinic acid, and chlorogenic acid. All of them were potent peroxynitrite scavengers (IC50 ≤ 1.88 μg/mL).Discussion and conclusion: The peroxynitrite scavengers were mainly distributed in the EtOAc fraction rather than the ether and BuOH fractions. The 70% MeOH extract exhibited a high peroxynitrite-scavenging activity. Through the validation, the present HPLC method was verified to be sufficiently sensitive, accurate, precise, and stable. Therefore, this method can be used for the quality control of A. iwayomogi.

Concurrent Analysis of Theanine, Caffeine, and Catechins Using Hydrophobic Selective C12 Stationary Phase

A fast reverse phase-high performance liquid chromatography-diode array detector (RP-HPLC-DAD) method has been developed and validated for simultaneous analysis of major bioactive constituents of tea. The collective separation of theanine, caffeine, and catechins has been achieved by using a new hydrophobic selective C12 stationary phase. The separation was done without any pre-column derivatization and using simple gradient solvent system of acetonitrile and water containing 0.01% TFA. We have achieved rapid separation of all these components within 16 min with very good peak resolution and sensitivity. The method also exhibits very good linearity with correlation coefficients between 0.9918 and 0.9999 for all the compounds. A comparative assessment study for the estimation of above-mentioned phytoconstituents from different solvent extracts as well as brewed infusions of green and black teas was also performed using this liquid chromatography (LC) method. The results of the comparative evaluation studies have showed that green tea extracts and brewed green tea infusions contain high yield of these phytoconstituents in comparison to black teas. The present HPLC method is quite applicable for biochemical analysis of wide range of tea samples including various types of teas, tea extracts, tea infusions, instant teas, ready-to-drinks (RTDs), and various other tea based value added food products and nutraceuticals and beverages.

Physical and chemical characterization of mefenamic acid in different pharmaceutical dosage forms and their stability studies using novel RP-HPLC method

A novel RP-HPLC method for the determination of mefenamic acid in raw material, different pharmaceutical forms, and in human serum for routine analysis, therapeutic purposes, and stability studies was developed and validated. This study is also a collection of studies performed on the physical and chemical aspects of mefenamic acid. The method developed constitutes mobile phase, acetonitrile:acetic acid:water (72.5:1:26.5, v/v/v) at pH 3 and mefenamic acid was monitored with UV detection at 279 nm, eluting out at 3.98 min. The present HPLC method was found to be linear (100–300 μg mL−1), accurate, and least time consuming with very good recovery. The limits of detection of the method were found to be 10 μg mL−1. This method was also used to study the stability profile of mefenamic acid in tablets for 5 years and suspension for 4 years. The excipients present in the formulation did not interfere with the assay. The present method was also compared with the UV–visible technique and it was observed that HPLC method is more precise, sensitive, and accurate.

Fluorometric determination of nitrite with 2,3-diaminonaphthalene by reverse phase HPLC under alkaline conditions

Introduction In this study, a simple and sensitive fluorometric HPLC method was described for the determination of nitrite, an indicator of nitric oxide production in biological samples. Methods Nitrite was reacted with 2,3-diaminonaphthalene (DAN) to form fluorescent 2,3-naphthotriazole (NAT). Because NAT has higher fluorescence intensity at alkaline conditions, a reverse phase HPLC method employing a polymer-based column was applied to separate NAT using an alkaline mobile phase system. Results NAT was well separated from DAN and other fluorescent substances with increased detection sensitivity under alkaline conditions. A linear relationship ( r = 0.999) between the fluorescence intensity of NAT and the concentration of nitrite was observed in the range from 0 to 800 nM with the detection limit of about 25 nM. Discussion The present HPLC method appears suitable for routine analysis of nitrite in crude biological samples, since the polymer-based column can withstand rigorous cleaning with either acid or base.

Quantification of astaxanthin in shrimp waste hydrolysate by HPLC

In the present study, a simple and rapid reversed-phase HPLC method for the determination of astaxanthin in shrimp waste hydrolysate has been developed and validated. The analytical procedure involves the direct extraction of astaxanthin from the lipid fraction with methanol. The analytical column, SS Exil ODS, was operated at 25C. The mobile phase consisted of a mixture of water:methanol:dichloromethane:acetonitrile (4.5:28:22:45.5 v/v/v/v) at a flow rate of 1.0 mL/min. Detection and identification were performed using a photodiode array detector (lambda(detection) = 476 nm). The proposed HPLC method showed adequate linearity, repeatability and accuracy.

인체 혈장 중 나돌올의 HPLC 분석법 검증 및 단회투여 후 약물동태 연구

A high-performance liquid chromatographic method was validated for quantitation of nadolol in human plasma. Nadolol and internal standard, pindolol, were extracted with tert-butyl methyl ether after addition of 10 M sodium hydroxide solution. The analytes were separated on a reverse phased C18 column using a mobile phase consisting of 0.05 M ammonium phosphate monobasic buffer, acetonitrile and methanol (81: 17:2 v/v/v) and detected using a fluorescence detector (excitation wavelength 230 nm, emission wavelength 294 nm). The method was specific and sensitive enough to detect as low as 3 ng/mL of nadolol in human plasma. Linear calibration range was 3-150 ng/mL with correlation coefficient greater than 0.999. The overall accuracy was in the range of 96.8 to 103% and precision C.V.(%) 7.30 to 12.2%. The recovery was approximately 100% and stability was confirmed during storage and sample preparation. The present HPLC method was successfully applied to study bioavailability after oral administration of 80 mg of nadolol in healthy Korean subjects. The mean was and of was reached at . The mean of nadolol was .

Analysis of plasma tocopherols α, γ, and 5-nitro-γ in rats with inflammation by HPLC coulometric detection

Reactive nitrogen oxide species (RNOS) have been implicated as effector molecules in inflammatory diseases. There is emerging evidence that gamma-tocopherol (gammaT), the major form of vitamin E in the North American diet, may play an important role in these diseases. GammaT scavenges RNOS such as peroxynitrite by forming a stable adduct, 5-nitro-gammaT (NGT). Here we describe a convenient HPLC method for the simultaneous determination of NGT, alphaT, and gammaT in blood plasma and other tissues. Coulometric detection of NGT separated on a deactivated reversed-phase column was linear over a wide range of concentrations and highly sensitive (approximately 10 fmol detection limit). NGT extracted from blood plasma of 15-week-old Fischer 344 rats was in the low nM range, representing approximately 4% of gammaT. Twenty-four h after intraperitoneal injection of zymosan, plasma NGT levels were 2-fold higher compared to fasted control animals when adjusted to gammaT or corrected for total neutral lipids, while alpha- and gammaT levels remained unchanged. These results demonstrate that nitration of gammaT is increased under inflammatory conditions and highlight the importance of RNOS reactions in the lipid phase. The present HPLC method should be helpful in clarifying the precise physiological role of gammaT.

RAPID HPLC ASSAY FOR LAMIVUDINE IN PHARMACEUTICALS AND HUMAN SERUM

ABSTRACT In the present study, a simple, sensitive, precise, and rapid HPLC method with UV detection for the analysis of lamivudine is developed and applied to the determination of anti HIV drug in commercial pharmaceutical dosage forms (tablets and oral solutions) and human serum. The gradient elution is performed with methanol:water (75 : 25, v/v) at a flow rate of 0.8 mLċmin−1, using a Spherisorb® C18 analytical column 150 × 4.6 mm, 5 µm. Deflazacort is used as internal standard. Absorbance is monitored at 265 nm. Total analysis time was ∼6 min. Data, with respect to precision and accuracy and limits of detection, are reported and discussed. The described method can be readily utilized for analysis of pharmaceutical products and pharmacokinetic studies as well.

Determination of free fatty acids in milk and dairy products by reversed-phase HPLC with fluorescence detection

A highly sensitive HPLC with fluorescence detection was developed for the determination of free fatty acids (FFAs) in milk and dairy products. For this purpose, the FFAs were extracted from small amounts of milk (1.0 mL), cheese (0.5 g) and butter (0.5 g) using Sep-Pak cartridge columns, and then derivatized to 9-anthrylmethyl esters with 9-anthryldiazomethane (ADAM). Good separations of the ADAM derivatives of 16 FFAs (C4-C18) that exist in milk, cheese and butter, which permitted quantitative estimation of their individual FFAs, were achieved by HPLC on a C18 column (Cadenza CD-C18, 150 x 3 mm i.d.) using gradient elution with methanol and water. The acid values calculated from the contents of individual FFAs were in good agreement with those obtained by the conventional titration method. These results demonstrate that the present HPLC method is simple, sensitive and precise, and could be utilized widely for determination of the FFAs in milk and dairy products.

Determination of Boronophenylalanine in Biological Samples Using Precolumn o-Phthalaldehyde Derivatization and Reversed-Phase High-Performance Liquid Chromatography

A reversed-phase high-performance liquid chromatographic method for the detection of boronophenylalanine is described. Determination was obtained by precolumn reaction of o-phthalaldehyde with a mixture of standard amino acids containing boronophenylalanine and separating the corresponding o-phthalaldehyde derivatives, using a Kromasil C-18, 250 × 4.6 mm, 5-μm particle size column, a step gradient with two buffers, a flow rate of 1.2 ml/min, a column temperature of 23°C, and fluorimetric detection (excitation and emission wavelengths of 330 and 430 nm, respectively). The use of such a method for assaying boronophenylalanine in biological samples was tested in neutralized perchloric acid blood and cerebral tissue extracts of rats treated with intracarotid administration of 300 mg/kg of body weight boronophenylalanine. Results of these experiments showed that the present HPLC method represents a valid alternative to currently available analytical techniques for assaying boronophenylalanine based on boron determination in terms of reproducibility, recovery, or sensitivity. Therefore, it is suggested that the present method may routinely be used in all preclinical and clinical studies in which quantification of circulating and tissue concentrations of boronophenylalanine is critical for the application of boron neutron capture therapy.

Quantitative analysis of derivatized proteins prepared with pyridyl disulfide-containing cross-linkers by high-performance liquid chromatography.

Determination of the introduced moieties into derivatized proteins is an essential step in the preparation and quality control of chemically defined immunoconjugates. For the derivatized proteins using pyridyl disulfide-containing cross-linkers such as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and 4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio)tolu ene (SMPT), the derivatization degree (ratio of pyridyl disulfide moieties to protein) has been traditionally determined by measuring the absorbance of both the derivatized protein and 2-thiopyridone (2-TP) released from the dithiothreitol (DTT) treatment (spectrophotometric method). This method, however, causes several problems including false high and low determinations of the protein and 2-TP, respectively, low selectivity, poor reproducibility, and relatively large amounts of sample consumption. A quantitative determination method of the derivatization ratios using bovine serum albumin derivatized with SPDP and SMPT as the model system has been developed. The concentration of protein and 2-TP released from the DTT treatment of derivatized proteins was determined directly without consideration of different reagents used and their concentrations. The present HPLC method was proved to be better in terms of accuracy, selectivity, and reproducibility with micro sample consumption. Moreover, this HPLC method can be directly applied to all derivatized proteins prepared with pyridyl disulfide-containing cross-linkers.

Preparation and Usefulness of Some Fluorogenic Substrates for Assay of Arginyl-tRNA-Protein Transferase by HPLC

A series of fluorescent substrates and products was prepared and evaluated for the assay of arginyl-tRNA-protein transferase (arginyltransferase) activity by HPLC. SinceN-aspartyl-N′-dansylamido-1,4-butanediamine (Asp(4)DNS) was the most suitable substrate of the compounds tested, which had a three-, four-, or five-methylene-chain interval between Asp or Glu and DNS, the following enzymatic studies were focussed on Asp(4)DNS and its product,N-arginylaspartyl-N′-dansylamido-1,4-butanediamine (ArgAsp(4)DNS). The apparentKmvalue for Asp(4)DNS was calculated to be 30 μM using a hydroxyapatite-treated arginyltransferase preparation from hog kidney, which was free from any enzyme that might decompose the two compounds. The present HPLC method was shown to be advantageous in reliability and sensitivity compared to the available isotope paper disk method using the hydroxyapatite-treated enzyme preparation and in applicability to crude samples examined using a DEAE-treated arginyltransferase preparation and 105,000gsupernatant (105S) from hog kidney. Stepwise elimination of Arg and Asp from ArgAsp(4)DNS was observed with the two crude enzyme solutions, and the elimination of Arg was suppressed by the addition of bestatin, suggesting the participation of certain aminopeptidases. Although Asp(4)DNS was decomposed significantly with 105S, an incubation-time-dependent linear elevation of ArgAsp(4)DNS was maintained for 5 min in the presence of bestatin. Furthermore, an addition–recovery test of the DEAE-treated enzyme preparation for the 105S assured accurate determination of arginyltransferase activity in the 105S under the tentatively established conditions. The present HPLC method, which permits the simultaneous determination of 4-dansylamidobutylamine, Asp(4)DNS, and ArgAsp(4)DNS, was advantageous in measuring arginyltransferase activity and detecting the presence of unfavorable enzyme(s) in samples to ensure accurate determination.