Gradient Elution Of Methanol Research Articles

Quantitative HPLC Analysis of Ascorbic Acid and Gallic Acid in Phyllanthus Emblica

A reversed-phase high-performance liquid chromatographic method has been developed and validated for simultaneous estimation of ascorbic and gallic acid in Phyllanthus emblica L. (Euphorbiaceae). A C18 column was used with a gradient elution of methanol and 0.1% (v/v) acetic acid in HPLC-grade water as mobile phase at a flow rate of 0.9 mL min -1 . UV detection was performed at 278 nm. The method was validated for accuracy, precision, linearity, specificity and sensitivity in accordance with International Conference on Harmonisation guidelines. Amounts of ascorbic and gallic acid detected in freeze-dried extract of the plant were 4.58% and 0.59%. Total run time was 50 min. ascorbic and gallic acid was eluted with retention times of 3.60 and 10.77 min respectively. Validation revealed that the method is specific, accurate, precise, reliable and reproducible. Calibration plots were linear over the concentration ranges 30–90 μg mL -1 for ascorbic acid and 5–15 μg mL -1 for gallic acid, respectively. Limits of detection were 1.42 and 1.56 μg mL -1 and limits of quantification were 4.32 and 4.73 μg mL -1 for ascorbic and gallic acid, respectively. Recovery was 99.37 % and 98.68% for ascorbic and gallic acid, respectively and the coefficient of variance was <2.0% for both. In negative ESI mode, the spectra showed the signals at m/z of 174.98 for ascorbic acid and m/z of 168.98 for gallic acid .The high percentage recovery and low coefficient of variation confirm the suitability of the method for simultaneous analysis of ascorbic and gallic acid in Phyllanthus emblica .

An Improved LC-DAD Method for Simultaneous Determination of Lutein, β-Carotene and Lycopene in Tomato and Its Products

An improved liquid chromatography method coupled with diode array detector was developed for the simultaneous determination of lutein, β-carotene and lycopene in tomato epicarp and products. The separation was achieved on a YMC C30 column with gradient elution of methanol and methyl tert-butyl ether. The improved method was simple, rapid, linear and sensitive for the simultaneous determination of the three carotenoids in tomato products.

Simultaneous analysis of six novel hallucinogenic (tetrahydrobenzodifuranyl)aminoalkanes (FLYs) and (benzodifuranyl)aminoalkanes (DragonFLYs) by GC-MS, LC-MS, and LC-MS-MS

Six novel hallucinogens classed as (tetrahydrobenzodifuranyl) aminoalkanes or (benzodifuranyl)aminoalkanes, which are known by the common names of “FLY” and “DragonFLY,” respectively, were synthesized. These compounds were simultaneously analyzed by gas chromatography (GC)-mass spectrometry (MS), liquid chromatography (LC)-MS, and LC-MS-MS. GCMS analysis of their free bases was not satisfactory for both mass spectral and chromatographic measurements, and thus trifluoroacetyl (TFA) derivatization was employed. However, it was found that the usual TFA derivatization procedure using trifluoroacetic anhydride caused dehydrogenation of FLYs to the corresponding DragonFLYs. Therefore, TFA derivatization of FLYs was reinvestigated; the presence of triethylamine could almost inhibit such dehydrogenation. LC separation of the analytes was successfully achieved by using a phenyl-type semimicro column with methanol gradient elution, while 1-(8-bromo-2,3,6,7-tetrahydrobenzo[1,2-b;4,5-b′]difuran-4-yl)-2-methylaminopropane (N-methyl-DOB-FLY) and 1-(8-bromo-2,3,6,7-tetrahydrobenzo[1,2-b;4,5-b′]difuran-4-yl)-2-aminopropane (DOB-FLY) were not separated on an octadecylsilica (ODS)-type column. Specific product ion spectra for all compounds were also obtained using LC-MS-MS, which enabled sensitive and reliable identification.

A novel rapid method for simultaneous determination of eight active compounds in silymarin using a reversed-phase UPLC-UV detector

A novel rapid chromatographic method based on utilization of UPLC column was developed for the analysis of eight active compounds in silymarin. The analysis was performed on a Waters Acquity UPLC system with an Acquity UPLC BEH C18 column (5 mm × 2.1 mm I.D., 1.7 μm) and a gradient elution of methanol and water containing 0.01% formic acid with a run time of 9 min, in which the retention time of the last analyte was 5.8 min. And all eight active compounds achieved complete separation. Comparison of system performance with conventional HPLC was made with respect to analysis time, efficiency and sensitivity. The results indicated that the type of column, the type of mobile phase and the modified addition were significant to the separation of isomeric compounds in herb extracts.

Optimization of extraction technique and validation of developed RP-HPLC-ELSD method for determination of terpene trilactones in Ginkgo biloba leaves

Terpene trilactones are potent and selective antagonists of platelet activating factor. The present study deals with standardization of efficient extraction method and validation of newly developed simple, sensitive and rapid reversed phase high performance liquid chromatographic method with evaporative light scattering detection (RP-HPLC-ELSD) method for the quantitative determination of ginkgolide A (GA), ginkgolide B (GB), ginkgolide C (GC), ginkgolide J (GJ) and bilobalide (BB) within 8 min in Ginkgo biloba leaf extract. The analysis was conducted on a Zorbax RP-C 18 column with gradient elution of methanol–water–tetrahydrofuran. The method was validated for accuracy, precession, limit of detection and quantification. The drift tube temperature of evaporative light scattering detector was set to 90 °C and nitrogen flow rate was 1.5 standard liter/min (SLM).

Simple and reliable methods for the determination of sixteen marker components for quality control of Daochi pill by HPLC coupled with diode array detection

Traditional Chinese medicine plays a very important role in the healthcare system of China and thus the quality control of medicinal herb products is of paramount concern. To establish a simple and effective high-performance liquid chromatography (HPLC) method to evaluate the quality of Daochi pill. Two HPLC methods were developed for the determination of 16 marker components in Daochi pills. In method A, the analytes were separated on a Lichrosorb C(18) column using a gradient elution of methanol and 1% aqueous phosphoric acid (pH 2.9, adjusted by triethylamine). In method B, the separation was achieved on an Agilent Eclipse Plus C(18) column using a gradient elution of methanol and 3% aqueous phosphoric acid (pH 2.0, adjusted by triethylamine) in a gradient elution mode. Methods were linear over the range 0.27-500 microg/mL (r(2) >or= 0.9995). Accuracy, precision and repeatability were all within the required limits. The mean recoveries measured at three concentrations were higher than 95% with RSD <or= 4% for the targets. The established methods are simple and can be used as tools for quality evaluation and control of this natural product.

Development and Validation of an RP-HPLC Method for Quantification of Cinnamic Acid Derivatives and Kaurane-Type Diterpenes inMikania laevigataandMikania glomerata

MIKANIA GLOMERATA and MIKANIA LAEVIGATA (Asteraceae) are medicinal plants popularly named 'guaco' in Brazil. The leaves of both species are used to treat respiratory diseases, with coumarin (CO) and kaurane-type diterpenes being regarded as the bioactive constituents. A new and simple RP-HPLC method was developed and validated for the simultaneous quantification of CO, O-coumaric (OC), benzoylgrandifloric (BA), cinnamoylgrandifloric (CA) and kaurenoic (KA) acids in the species. Optimal separation was achieved with an alternating gradient elution of methanol and acetonitrile and detection was carried out by DAD at three different wavelengths: 210 nm for CO, OC, KA; 230 nm for BA; and 270 nm for CA. The extracts showed good stability during 42 hours under normal laboratory conditions (temperature of 23 +/- 2 degrees C). The standard curves were linear over the range 0.5 - 5.0 microg (CO), 0.25 - 4.0 microg (OC), 1.0 - 8.0 microg (BA), 0.5 - 3.0 microg (CA) and 0.8 - 12.0 microg (KA), with R(2) > 0.999 for all compounds. The method showed good precision for intra-day (RSD < 4.6 %) and inter-day assays (RSD < 4.4 %). The recovery was between 99.9 and 105.3 %, except for CO and OC in M. glomerata (73.2 - 91.6 % and 86.3 - 117.4 %, respectively). The limits of quantification and detection were in the range of 0.025 - 0.800 microg and 0.007 - 0.240 microg. The method was tested for new and old columns, temperature variation (26 and 28 degrees C) and by different operators in the same laboratory. The method was successfully applied to samples of both species.

Simultaneous Determination of Eight Phenylethanoid Glycosides in Different Species of the Genus Cistanche by High Performance Liquid Chromatography

Although Traditional Chinese Medicine has been used for several thousands of years in clinical practice, the efficacy, quality, and safety of many medical herbs remain uncertain. There is an increasing need for improving analytical methods that can be used for identification of active components and quality control of traditional Chinese medicine. In the present study, a high performance liquid chromatographic (HPLC) method was developed to simultaneously determine eight phenylethanoid glycosides in different species of the genus Cistanche, namely acteoside, 2′-acetylacteoside, cistanoside A, cistanoside C, cistanoside F, echinacoside, isoacteoside, and tubuloside A. The HPLC assay was performed on an Akasil C18 column with a gradient elution of methanol and 0.1% aqueous methanoic acid for 70 min. The detection wavelength was 330 nm. All of the compounds showed good linearity (R2 ≥ 0.9991). The method was reproducible with intra- and inter-day variations of less than 5%. The recovery of the assay was in the range of 94.43–105.44%. The method was successfully applied to the quantification of all the eight constituents in the test samples. The results indicate that the developed assay can be considered as a suitable method for the quality control of the genus Cistanche.

Simultaneous quantitation of aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine and benzoylhypaconine in human plasma by liquid chromatography–tandem mass spectrometry and pharmacokinetics evaluation of “SHEN-FU” injectable powder

A rapid, specific and sensitive liquid chromatography–tandem mass spectrometry (LC/MS/MS) method was developed for simultaneous quantitation of six Aconitum alkaloids, i.e. aconitine (AC), mesaconitine (MA), hypaconitine (HA), benzoylaconine (BAC), benzoylmesaconine (BMA) and benzoylhypaconine (BHA) in human plasma collected from 18 healthy volunteers after intravenous drop infusion of “SHEN-FU” injectable powder in three different dosages. Lappaconitine was selected as the internal standard (IS). LC/MS/MS system coupled with an electrospray ionization (ESI) source was performed in multiple-reaction monitoring (MRM) mode. The transitions of the Aconitum alkaloids executed as following: m/ z 646.3 → 586.0 for AC; m/ z 632.4 → 573.1 for MA; m/ z 616.2 → 556.1 for HA; m/ z 604.2 → 104.8 for BAC; m/ z 590.1 → 104.8 for BMA; m/ z 574.1 → 104.8 for BHA; m/ z 585.2 → 161.8 for IS. Sample preparation was performed with solid-phase extraction (SPE) on a 1 mL HLB cartridge prior to analysis. The separation was applied on a Waters C 18 column (1.7 μm, 2.1 mm × 100 mm) and a gradient elution of methanol and 0.1% formic acid–water was used as mobile phase. The retention time was less than 4.5 min. The concentrations ranged from 0.1 to 1000 ng/mL for all six Aconitum alkaloids and showed a good linearity with the correlation coefficient ( r 2) >0.995. The validated method was employed to simultaneous quantitation and successfully used for the first time for the pharmacokinetic evaluation of the six Aconitum alkaloids after intravenous drop administration of “SHEN-FU” injectable powder in phase I clinical trial.

Structural elucidation of in vitro metabolites of emodin by liquid chromatography–tandem mass spectrometry

Liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was employed to investigate the in vitro metabolism of emodin. Emodin was incubated with rat liver microsomes in the presence of a NADPH-generating system, followed by extraction with ethyl acetate. After separation on a reversed-phase C18 analytical column with a linear gradient elution of methanol and 0.1% formic acid in water, negative electrospray ionization tandem mass spectrometry experiments were performed. As a result, the parent drug and its six metabolites were detected from rat liver microsomal incubations. The identification of the metabolites and elucidation of their structure were performed by comparing the changes in molecular masses (DeltaM), retention times and MS(2) spectral patterns of metabolites with those of parent drug. Besides three mono-hydroxylated metabolites (omega-hydroxyemodin, 2-hydroxyemodin, 4-hydroxyemodin), three other metabolites were identified, which were emodic acid, 3-carbomethoxy-6-methoxy-1,8-dihydroxyanthraquinone and physcion, respectively.

Separation of quercetin, sexangularetin, kaempferol and isorhamnetin for simultaneous HPLC determination of flavonoid aglycones in inflorescences, leaves and fruits of three Sorbus species

A reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated for the simultaneous determination of four flavonol aglycones (quercetin, QU; sexangularetin, SX; kaempferol, KA; isorhamnetin, IS) in hydrolyzed extracts from different plant parts of Sorbus aucuparia L., Sorbus aria (L.) Crantz. and Sorbus intermedia (Ehrh.) Pers. Separation of the four compounds was accomplished on a C18 Lichrosphere 100 column (5 μm, 250 mm × 4.6 mm, i.d.) with a methanol gradient elution and recorded at 370 nm. The high resolution of critical bands – SX, KA and IS – was achieved with retention of the last peak (IS) in 19.5 min. The equilibration of the standard mixture by addition of HCl to an acid concentration equal that of hydrolyzed extracts injected was found to be necessary when minimizing calibration error. The correlation coefficients of all the calibration curves showed good linearity ( r > 0.9991) over the test range. The relative standard deviation of the method was less than 2.8% for intra- and inter-day assays, and the average recoveries were between 95.5 and 102.5%. High sensitivity was demonstrated with detection limits between 0.050 and 0.085 μg/ml. The level of total aglycones was found to be in the range of 687–1515 mg/100 g of dry weight in the inflorescences, 424–1078 mg/100 g in the leaves and 20–60 mg/100 g in the fruits depending on the Sorbus species.

A selective fluorescence reaction for peptides and chromatographic analysis

A novel and selective fluorescence reaction is proposed for the quantitative determination of peptides by reversed-phase liquid chromatography (RPLC). A single fluorescent product was formed when a peptide was heated at 120 °C for 20 min in a neutral aqueous medium (pH 7.0) containing catechol, sodium periodate, and sodium borate. The fluorescent products of four peptides such as Leu-Gly, Ala-Leu-Gly, Tyr-Gly-Gly-Phe-Leu, and Leu-Leu-Leu were easily separated on a reversed-phase column by gradient elution of methanol in a mobile phase containing sodium borate (pH 7.0), and then quantitatively detected by fluorimetry. The lower limits (S/N = 3) of the detection for the tested peptides were 0.5–1.0 pmol per an injection volume (40 μl). In addition, the fluorescent products of phenylalanine amide and Leu-Leu-Leu were identified by electrospray ionization-time of flight-mass spectrometry (ESI-TOF/MS) for the elucidation of their chemical structures.

Separation and determination of polyethylene glycol fatty acid esters in cosmetics by a reversed-phase HPLC/ELSD

A comprehensive analytical method was established for the separation of polyethylene glycol (PEG) stearates according to the distribution of ethylene oxide (EO) and subsequent determination of the surfactants in cosmetic samples by using a high-performance liquid chromatography–evaporative light scattering detection. Separation of the PEG stearates comprising approximately up to 82 EO adducts was performed on a reversed-phase YMC-Pack C 8 column using water–acetonitrile gradient elution. The PEG oligomers were separated in order of the increasing number of EO adducts. Quantitation of the PEG fatty acid esters, which was separated as single peak per each component, was performed by chromatography on a reversed-phase Wakosil 10 C 18 column using water–methanol gradient elution. The standard curve to quantify the PEG stearates was constructed by the log–log plot, which showed good linearity with the correlation coefficients ( R 2) 0.998 and more. Working range, repeatability, limit of detection and recovery were acceptable for analysis of the surfactants in cosmetic products. The analytical methods were applied to characterize the PEG stearates according to the EO distributions, then to quantify the surfactants in cosmetic products.

Simultaneous determination of seven major diterpenoids in Pseudolarix kaempferi by high-performance liquid chromatography DAD method

A reversed phase high-performance liquid chromatography method was established for the first time to simultaneously qualify the seven major diterpenoids in Pseudolarix kaempferi, namely pseudolaric acid B O-β- d-glucopyranoside ( 1), pseudolaric acid C 2 ( 2), pseudolaric acid C 1 ( 3), deacetylpseudolaric acid A ( 4), pseudolaric acid A O-β- d-glucopyranoside ( 5), pseudolaric acid B ( 6) and pseudolaric acid A ( 7). The optimal conditions of separation and detection were achieved on an Inertsil ODS-3 column with gradient elution of methanol and 0.5% aqueous acetic acid (v/v) at the flow rate of 0.6 ml min −1 within 40 min and detection wavelength set at 262 nm. All calibration curves showed good linear regression ( r 2 > 0.9999) within test ranges. This method provided good accuracy with recoveries in the range of 94.3–106.1% and good precision with R.S.D.s of repeatability and intermediate precision less than 0.57% and 4.67%, respectively. The method was successfully applied to qualitative and quantitative determination of 20 P. kaempferi among the 54 samples collected from different areas. The results revealed that the commercial crude drugs were seriously confused and the developed HPLC assay could be used as a suitable qualitative and quantitative determination method for P. kaempferi.

Simultaneous determination of 12 major constituents in Forsythia suspensa by high performance liquid chromatography—DAD method

A high performance liquid chromatographic method was developed to simultaneously determine the 12 major constituents of Forsythia suspensa, namely R-suspensaside, S-suspensaside, S-suspensaside methyl ether, (+)-pinoresinol-β- d-glucoside, forsythiaside, (+)-epipinoresinol-4′- O-glucoside, suspensaside A, rutin, phillyrin, (+)-pinoresinol, (+)-epipinoresinol and phillygenin. The HPLC assay was performed on a Zorbax XDB C 18 column with gradient elution of methanol and 0.3% aqueous acetic acid within 55 min. The detection wavelength was 280 nm. All the compounds showed good linearity ( r 2 > 0.9998). The method was reproducible with intra- and inter-day variation less than 3%. The recovery of the assay was in the range of 91.2–104.9%. The method was successfully applied to the quantification of 12 constituents in 33 F. suspensa samples. The results indicated that the developed assay could be considered as a suitable quality control method for F. suspensa.

Method optimization for the determination of four mercury species by micro-liquid chromatography–inductively coupled plasma mass spectrometry coupling in environmental water samples

A method based on the coupling microHPLC–microneb-ICPMS has been developed for Hg(II), MeHg +, EtHg + and PhHg + species. Gradient elution using methanol and l-cysteine at pH 3.0 allowed the chromatographic separation of all species in less than 13 min (total analysis time 15 min). The direct coupling of microLC to ICPMS through a Micromist nebulizer permits the analysis of environmental water without sample pretreatment and derivatization steps. Nebulizer type, organic modifier and column length were the main parameters tested. The methanol content and pH of the mobile phase greatly affected the retention time and sensitivity of the method. Key factors to obtain high signal to noise ratio, at concentrations below 1 μg L −1, were found to be the nebulization step and traces of Hg present in the complexing agent. A detailed optimization of carrier and make up gas flow rates have enabled the nebulization of the methanol gradient elution with good mass transport efficiency, low organic solvent loading into the plasma and excellent precision. The performance of the microHPLC–microneb-ICPMS method developed was evaluated on a surface water sample filtered (0.22 μm) and spiked with 0.5 μg L −1 (as Hg) of each species. Precision (R.S.D., n = 6) for all species of Hg varied from 0.5 to 2.1%. Detection limit, defined as three times the standard deviation ( n = 6), ranged from 8 ng L −1 for EtHg + to 32 ng L −1 for PhHg + and was noticeably lower than those reported in previous LC-based methods. Accuracy was suitable with recoveries ranging from 85 to 100% when tested at two levels (0.5 and 10 μg L −1) in groundwater samples. Recovery was matrix affected when water samples of high salinity (depurated wastewater and seawater) were used.

Qualitative and quantitative determination of nucleosides, bases and their analogues in natural and cultured Cordyceps by pressurized liquid extraction and high performance liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI–MS/MS)

A simple and rapid pressurized liquid extraction (PLE) and high performance liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI–MS/MS) method was developed for qualitative and quantitative determination of nucleosides, bases and their analogues in natural and cultured Cordyceps. The samples were extracted using PLE. The separation was achieved on a ZORBAX Eclipse XDB-C 18 column with gradient elution of methanol and 5 mM aqueous ammonium acetate as mobile phase. Target compounds were identified by characterizing their product ions, precursor ions and retention times. Quantitative analysis of investigated compounds were performed using time programmed selective ion monitoring (SIM) or selective reaction monitoring (SRM) with 10 segments in positive (negative for uridine) ion mode. The results showed that 43 bases, nucleosides and their analogues were detected in Cordyceps, of these 16 compounds were identified. The simultaneous determination of seven nucleosides and six bases in Cordyceps was achieved using PLE and HPLC–ESI–MS/MS method described above, which afforded good linearity, selectivity, precision, recovery, short analysis time as well as LOD and LOQ in the ng/ml range.

Quantitation of the niacin metabolites 1-methylnicotinamide and l-methyl-2-pyridone-5-carboxamide in random spot urine samples, by ion-pairing reverse-phase HPLC with UV detection, and the implications for the use of spot urine samples in the assessment of niacin status

A simple ion-pairing reverse-phase HPLC method, with UV diode array detection, was developed and validated for quantitation of the urinary niacin metabolites 1-methylnicotinamide and l-methyl-2-pyridone-5-carboxamide in a single run. Urine samples were purified using a polymer-based mixed mode anion exchange reverse-phase cartridge. Analysis was performed on a reverse-phase C18 column, using a methanol gradient elution system, containing phosphate buffer pH 7.0, 1-heptanesulphonic acid as the ion-pairing agent and trimethylamine as a modifier. The assay was applied to the measurement of the niacin status of two subjects using spot urine samples. The samples were collected over 4 consecutive days and at four time points during 1 day. Status, expressed as the concentration ratios (2-PYR or 1-MN)/creatinine and 2-PYR/l-MN, varied within and between days and was least for fasting samples. This work illustrates the potential of spot urine sampling for niacin status assessment, but highlights the need for further validation prior to its use in field nutritional surveys.

Determination of thiamine in blood serum and urine by high-performance liquid chromatography with direct injection and post-column derivatization

Thiamine (vitamin B 1) was determined in human serum and urine by HPLC with fluorimetric detection of its oxidation product, thiochrome. The samples were injected directly into the chromatographic system without previous treatment or dilution. A column filled with an ultra-high molecular weight surface-modified polyethylene (PE) was able to separate matrix components from analyte and also to allow a good chromatographic resolution of thiamine. The interaction of thiamine, thiocrome and both matrices (serum and urine) with PE was studied off- and on-line to determine the optimal procedure for vitamin B 1 determination. When carried off-line, matrix adsorption yield was 49 mg serum proteins/g polymer and components of 1000 μl urine/g polymer. In an on-line arrangement, the yield dropped to 10 mg/g and 150 μl/g, respectively. The matrix/analyte separation was carried out in an on-line procedure on a 50×4.6-mm, 25-μm PE column, using a water–sodium phosphate–methanol gradient elution. Part of the matrix was eluted within the first 2 min and thiamine after 3.8 min. The rest of the matrix retained on the column was eluted after thiamine at the last step of the gradient elution. Analysis time was 12 min. The within-day and day-to-day precision gave C.V. varying from 3.6% to 14.5% and recoveries from spiked samples were in the range of 84.8–98.8%.

Assay of leucine aminopeptidase activity in vitro using large-pore reversed-phase chromatography with fluorescence detection

A chromatographic method for determination of leucine aminopeptidase (LAP) activity in complex matrices is described. l-Leucine-β-naphthylamide was used as the substrate and its hydrolytic product, β-naphthylamine, was monitored by fluorescence at 280 nm excitation and 400 nm emission wavelengths. Under optimized conditions, the components in the incubation mixture were baseline separated and eluted out of a large-pore (300 Å) reversed-phase C 4 column (RPC 4) within 15 min with a non-linear gradient elution of methanol (0.05% (v/v) trifluoroacetic acid additive). The detection limit of the hydrolytic product reached 0.35 pmol at three time signal-to-noise (S/N) ratio with 5 μl sample injection. The method showed a wide dynamic range for quantitation of both the hydrolytic product (10 ng/ml to 80 μg/ml) and LAP (0.1–46.0 μg/ml) with correlation coefficient larger than 0.998 and reproducibility <3 and 10% R.S.D. ( n=3), respectively. A fairly broad range of incubation time could be selected within 1 h. The LAP activities and concentrations in rabbit serum, tears, and mouse lens homogenates were determined to be 41.8 (0.3 mg/ml), 2.8 (40.0 μg/ml), and 1.6 pmol/(μl min) (17.5 μg/ml), respectively, with reproducibility of 2–9% R.S.D. ( n=3) and intra- and inter-day variation for the retention time of the hydrolytic product being <1% R.S.D. ( n=3). The results indicate that the present method is rapid and sensitive as compared to the conventional one.