Method for the determination of beta-carotene in supplements and raw materials by reversed-phase liquid chromatography: single laboratory validation

A single laboratory validation (SLV) study was conducted for a liquid chromatography (LC) method for the determination of total and all-trans-beta-carotene in a variety of dietary supplements, including multivitamin tablets, softgels, capsules, and beadlet raw materials. Extraction variants were developed for the different types of supplements tested based upon the supplement type and level of beta-carotene. Water dispersible formulations such as powders, emulsions, tablets, and capsules were enzymatically digested with protease and extracted with dichloromethane-ethanol. Oily suspensions were directly dissolved in dichloromethane-ethanol. After appropriate dilution or concentration, the extracts were chromatographed by using either a reversed-phase C18 column or, in products containing high amounts of alpha-carotene, a reversed-phase C30 column. The LC systems provided linear responses in the range of 0.1-50 microg beta-carotene/mL. The main geometrical isomers of beta-carotene (all-trans, 9-cis, 13-cis, and 15-cis) were well separated from each other and from other carotenoids such as a-carotene, cryptoxanthin, lutein, lycopene, and zeaxanthin. Duplicate determinations of total beta-carotene performed by 2 technicians in 8 different test materials on 5 different days resulted in relative standard deviations of 1.2-4.4%. Recoveries determined for supplements and beadlet raw material spiked with beta-carotene levels of 10 microg to 100 mg/test portion and 0.2-40%, respectively, ranged from 97.5 to 102.1%. On the basis of the accuracy, precision, and recovery results from the SLV study, the method is suggested for a collaborative study on the determination of total and all-trans-beta-carotene in dietary supplements.     

Simultaneous determination of all-trans-retinoic acid, beta-carotene, and vitamin A in galenic preparations by liquid chromatography

The concentrations of vitamin A, beta-carotene, and all-trans-retinoic acid in oral preparations were determined in a single analysis by a method based on isocratic, reversed-phase liquid chromatography (LC). The LC system consisted of a C18 column, a mobile phase of acetonitrile, dichloromethane, methanol, and water and a UV detector set at 330 nm. The linearity ranges were 25-250 ng/mL for trans-retinoic acid and vitamin A, and 100-1,000 ng/mL for beta-carotene. This LC method for the determination of retinoids is simple, precise, and accurate. No extraction procedure is required before the chromatographic analysis; only a suitable dilution is necessary. The method proved to be reliable, fast, and economical. Furthermore, this method is indicative of stability, because it allows for the determination of degradation products such as 13-cis-retinoic acid.     

Determination of cholecalciferol (vitamin D3) in selected foods by liquid chromatography: NMKL collaborative study

Results are presented from an NMKL (Nordic Committee on Food Analysis) collaborative study of a method for the determination of cholecalciferol (vitamin D3) in foods. The method is based on the addition of an internal standard (vitamin D2), followed by saponification and extraction with n-heptane. The fraction that contains vitamin D2/D3 is separated by preparative normal-phase liquid chromatography (LC), and the analytes are determined by reversed-phase LC with UV detection at 265 nm. The method was tested by 8 participating laboratories. In this study 6 different matrixes were analyzed for cholecalciferol content: milk, liquid infant formula (gruel), cooking oil, margarine, infant formula, and fish oil. The contents varied from 0.4 to 12 microg/100 g. Three matrixes (milk, gruel, and margarine) were fortified with vitamin D3. In the other matrixes, vitamin D3 was added at 3 different levels at the Swedish National Food Administration. The milk was analyzed as a blind duplicate, whereas the other matrixes were analyzed as split-level pairs. The recoveries from the samples with vitamin D3 added varied from 93 to 102%. The repeatability relative standard deviation (RSDr) values for accepted results varied between 2.2% (fish oil) and 7.4% (cooking oil), whereas the reproducibility relative standard deviation (RSD(R)) values varied between 6.8% (margarine) and 24% (cooking oil).     

Quantitative analyses of beta-carotene and retinol in serum and feces in support of clinical bioavailability studies

Among more than 50 provitamin carotenoids, beta-carotene is the most metabolically active source of retinol. Despite diets rich in fruits and vegetables containing beta-carotene, vitamin A deficiency is the leading cause of blindness and childhood mortality in developing countries. In addition, the uncertainty of beta-carotene bioconversion into vitamin A suggests that new data are needed to update the nutritional guidelines in developed countries. Previously, we reported the development of a carotene/retinol plateau isotopic enrichment method (CarRet PIE) for the determination of beta-carotene bioavailability and bioconversion into retinol, which utilizes positive ion atmospheric pressure chemical ionization (APCI) liquid chromatography/mass spectrometry (LC/MS). While seeking to validate the CarRet PIE using a mass balance approach requiring fecal measurements of beta-carotene and retinol, interference was encountered that required substantial modifications of the LC/MS assay. Here we report a new LC/MS assay that is based on the detection of molecular anions of beta-carotene using negative ion APCI with a reversed-phase C30 column for HPLC separation. Sample preparation required saponification to eliminate interfering triglycerides. The limit of detection (LOD) of beta-carotene was 0.25 pmol calculated on the basis of an injection of 20 microL of 0.0125 microM beta-carotene, and the limit of quantitation (LOQ) was 1.0 pmol based on the injection of 20 microL of 0.050 microM beta-carotene. The linear range was 1.1 to 2179 pmol on-column. The wide linear range and low LOD and LOQ of this assay facilitated the sensitive and selective quantitative analysis of beta-carotene in both serum and fecal samples in support of an on-going clinical investigation of beta-carotene bioavailability and bioconversion into vitamin A.     

An interlaboratory-verified method for the determination of vitamins A and E in milk- and soy-based infant formula by liquid chromatography with matrix solid-phase dispersion extraction

An interlaboratory-verified, liquid chromatographic (LC) method is presented for determination of all-racemic alpha-tocopheryl acetate and retinyl palmitate in infant formula. The extraction procedure uses matrix solid-phase dispersion. A sample is mixed with C18, and the mixture is packed into a reservoir and eluted with selective solvents to extract the analytes. After evaporation and filtration, the sample extract is injected directly into a normal-phase LC system with fluorescence detection. All-racemic alpha-tocopheryl acetate and retinyl palmitate are quantitated isocratically with a mobile phase of hexane containing isopropanol at 0.2% (v/v) and 0.125% (v/v), respectively. A nonfortified zero control reference material (ZRM) was spiked at 5 levels, with 5 replicate analyses of 1/2x, x, 2x, 4x, and 16x where "x" represents the minimum levels of 250 IU/100 kcal (vitamin A) and 0.7 IU/100 kcal (vitamin E) as specified in Title 21 of the Code of Federal Regulations, part 107.100. Recoveries of retinyl palmitate ranged from 83.8 to 107%, and those of all-racemic alpha-tocopheryl acetate ranged from 87.7 to 108%. Two additional laboratories analyzed the ZRM samples at 4 spiking levels with 6 replicates. Recoveries of retinyl palmitate and all-racemic alpha-tocopheryl acetate ranged from 92.2 to 104% and from 91.7 to 101%, respectively, in the second laboratory. Recoveries of retinyl palmitate and all-racemic alpha-tocopheryl acetate ranged from 85.3 to 97.0% and from 86.6 to 110%, respectively, in the third laboratory. Relative standard deviations for all 3 laboratories ranged from 0.2 to 7.5% with an average of 2.9%. In addition, each laboratory analyzed a commercial milk- and commercial soy-based infant formula. Excellent agreement in results was obtained between the 3 laboratories for vitamins A and E in all matrixes.     

Quantitative determination of folic acid in multivitamin/multielement tablets using liquid chromatography/tandem mass spectrometry

Two different isotope-dilution liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods for the quantitative determination of folic acid (FA) in multivitamin/multielement tablets are reported. These methods represent distinct improvements in terms of speed and specificity over most existing microbiological and chromatographic methods for the determination of FA in dietary supplements. The first method utilizes an aqueous/organic-based extraction solvent combined with positive-ion mode LC/MS/MS detection of protonated [M + H]+ FA molecules and the second method utilizes a pure aqueous-based extraction solvent combined with negative-ion mode LC/MS/MS detection of deprotonated [M - H]- FA molecules. The LC/MS/MS methods exhibit comparable linear dynamic ranges (> or =3 orders of magnitude), limits of detection (0.02 ng on-column) and limits of quantification (0.06 ng on-column) for FA. Two methods employing different extraction and different MS detection modes were developed to allow method cross-validation. Successful validation of each measurement procedure supports the use of either method for the certification of FA levels in dietary supplements. The accuracy and precision of each measurement procedure were evaluated by applying each method to the quantitative determination of FA in a NIST standard reference material (NIST SRM 3280 multivitamin/multielement tablets). The FA measurement accuracy for both methods was > or =95% (based on the manufacturer's assessment of the FA level in SRM 3280) with corresponding measurement precision values (% RSD) of approximately 1%.     

Method for the determination of Aconitum alkaloids in dietary supplements and raw materials by reversed-phase liquid chromatography with ultraviolet detection and confirmation by tandem mass spectrometry: single-laboratory validation

A study of single-laboratory validation (SLV) of a reversed-phase liquid chromatography (RP-LC) method was conducted for the determination of diester-diterpene Aconitum alkaloids, viz., aconitine, mesaconitine, and hypaconitine, in a variety of dietary supplements, including single- and multiple-ingredient dry powder extracts, pills, capsules, and raw materials. The Aconitum alkaloids in the samples were extracted by diethyl ether in the presence of ammonia. After cleanup with solid-phase extraction to remove the matrix interferences, the alkaloids were determined by RP-LC with UV detection at 235 nm, and the results were confirmed by tandem mass spectrometry. The linear responses for aconitine, mesaconitine, and hypaconitine based on the present LC system ranged from 0.5 to 200 microg/mL. Relative standard deviations of 2.0 to 6.9% were obtained from duplicate analysis of 6 test materials of different matrixes for the 3 Aconitum alkaloids performed by 2 analysts on 5 different days. The recoveries determined for supplements and raw materials spiked with 3 Aconitum alkaloids at levels of 2.5-10 microg/g were in the range of 86-99%. In view of the attainment of satisfactory results for accuracy, precision, and recovery in the SLV study, it is recommended that the method validation process proceed to a collaborative study.     

Determination of St. John's wort components in dietary supplements and functional foods by liquid chromatography

St. John's wort (Hypericum perforatum L.) preparations, a top-selling botanical dietary supplement used primarily as an antidepressant, has recently been used as an ingredient in some food products sold as functional foods. A rapid extraction technique followed by a liquid chromatographic (LC) method was developed to determine 4 characteristic bioactive compounds (pseudohypericin, hypericin, hyperforin, and adhyperforin) from St. John's wort in dietary supplements and functional foods to which it was added. Solid samples, including dried leaf/flower mixture, dietary supplement capsules, tea bags, puff and snack bar, were extracted with methanol by sonication. Noncarbonated, fruit-flavored drinks were centrifuged and mixed with methanol. Compounds were then determined by isocratic, reversed-phase LC with UV detection at 2 wavelengths and further identified or confirmed by photodiode array spectra and LC/mass spectrometry. Within-laboratory method variations (% RSD) were satisfactory. Very low amounts, if any, of the 4 components were found in drink and puff samples, and none was found in the snack bar. The methods developed provide a useful means for the determination of St. John's wort components in dietary supplements and functional foods.     

Determination of phenolic compounds in dietary supplements and tea blends containing Echinacea by liquid chromatography with coulometric electrochemical detection

Analytical methodologies with ultrasonic extraction and liquid chromatography (LC) were developed for the determination of phenolic compounds in dietary supplements containing Echinacea. The phenolic compounds determined by these methods included caftaric acid, chlorogenic acid, cynarin, echinacoside, and cichoric acid. Samples from tablets, capsules, and bags of tea blends were extracted by sonication for < or = 30 min with methanol-water (60 + 40). The extracts were centrifuged and filtered, and the filtrates were diluted and analyzed by LC using a reversed-phase column and coulometric electrochemical (EC) detection. The mobile phase was acetonitrile-ammonium formate buffer, pH 3.5 (15.3 + 84.7) containing tetrabutyl ammonium hydrogen sulfate as an ion-pairing reagent. Extraction conditions (e.g., composition of the extraction solvent and sonication time) were optimized for different types of samples. Intra- and interday analytical variations were determined, and intraday analyses were performed by 2 independent analysts using 2 different LC systems. Results were generally comparable. The LC method with EC detection showed better sensitivity and selectivity when compared with LC with ultraviolet detection, although results were similar for the 2 methods for major compounds, i.e., caftaric acid, echinacoside, and cichoric acid. The identities of these major compounds found in samples were confirmed by LC/electrospray ionization mass spectrometry.     

Determination of beta-carotene in supplements and raw materials by reversed-phase high pressure liquid chromatography: collaborative study

Twelve laboratories representing 4 countries participated in an interlaboratory study conducted to determine all-trans-veta-carotene and total beta-carotene in dietary supplements and raw materials. Thirteen samples were sent as blind duplicates to the collaborators. Results obtained from 11 laboratories are reported. For products composed as softgels and tablets that were analyzed for total beta-carotene, the reproducibility relative standard deviation (RSDR) ranged from 3.35 to 23.09% and the HorRat values ranged from 1.06 to 3.72. For these products analyzed for trans beta-carotene, the reproducibility relative standard deviation (RSDR) ranged from 4.28 to 22.76% and the HorRat values ranged from 0.92 to 3.37. The RSDr and HorRat values in the analysis of a beadlet raw material were substantial and it is believed that the variability within the material itself introduced significant variation in subsampling. The method uses high pressure liquid chromatography (LC) in the reversed-phase mode with visible light absorbance for detection and quantitation. If high levels of alpha-carotenes are present, a second LC system is used for additional separation and quantitation of the carotene species. It is recommended that the method be adopted as an AOAC Official Method.     

Determination of retinoids in galenicals by column liquid chromatography with fluorescence and diode-array detection

Simple and rapid reversed-phase gradient column liquid chromatography (LC) with fluorescence detection at different wavelengths was developed for the simultaneous analysis of all-trans, 13-cis, 9-cis retinoic acids, vitamin A palmitate and beta-carotene in galenicals. The assay results agreed with those obtained by an LC method with diode-array UV detection. A post-column on-line photochemical reactor (irradiation at 254 and 366 nm) was inserted between the LC column and the fluorescence detector to enhance the performance of the method. Two fluorescence spectra (photoreactor on and off) were obtained for each analyte which proved useful for the unambiguous identification of the various analytes.     

Improved liquid chromatographic method for the analysis of photosynthetic pigments of higher plants

The paper presents an improved reversed-phase LC method for the separation of the pigments from green leaves. A good separation of carotenoids and of their cis- and trans-isomers was achieved, especially for the separation of trans-lutein, zeaxanthin, cis-lutein, which are usually not well separated. No perfect separation of alpha-carotene, beta-carotene and pheophytin a was possible, but conditions for a perfect coelution of pheophytin a with either beta-carotene or alpha-carotene were established. Simultaneous equations allowing the determination of pheophytin a and alpha-carotene or pheophytin a and beta-carotene are also given.     

Determination of retinyl palmitate (vitamin A) in fortified fluid milk by liquid chromatography: collaborative study

A liquid chromatographic (LC) method was developed for fast and simple measurement of retinyl palmitate (vitamin A) in fortified milk. Retinyl acetate internal standard was added to a test portion of milk followed by extraction into hexane. The hexane extract was analyzed by LC using a normal-phase silica gel column equilibrated with mobile phase (conditioned hexane-isopropanol, 99.85 + 0.15, v/v) about 1 h before injections. The retinyl palmitate concentration was calculated by using a relative response factor determined with calibration standards. In the collaborative study, 11 laboratories analyzed 13 pairs of fluid milk materials in blind duplicate. Twelve of the materials were composed of skim milk (< 0.5% fat), 1% fat milk, 2% fat milk, and 1% fat chocolate milk. Each material was fortified at 3 concentrations of retinyl palmitate of approximately 581 microg/L (1000 IU/qt), 1163 microg/L (2000 IU/qt), and 2236 microg/L (4000 IU/qt). The 13th material, unfortified skim milk, served as a matrix blank. Repeatability standard deviations (RSDr) without outliers ranged from 1.5 to 5.7% and reproducibility standard deviations (RSDR) without outliers ranged from 5.0 to 22.7%. cis-Isomers co-eluted with the predominant trans-retinyl palmitate isomer and were included in the results reported by all the collaborative laboratories. Endogenous long-chain esters from milk fat were also measured with the retinyl palmitate additive. The Study Director recommends that this method for determination of retinyl palmitate in fluid milk by LC be adopted First Action.     

Determination of aristolochic acid in botanicals and dietary supplements by liquid chromatography with ultraviolet detection and by liquid chromatography/mass spectrometry: single laboratory validation confirmation

The presence of aristolochic acid in some dietary supplements is a concern to regulators and consumers. A method has been developed, by initially using a reference method as a guide, during single laboratory validation (SLV) for the determination of aristolochic acid I, also known as aristolochic acid A, in botanical species and dietary supplements at concentrations of approximately 2 to 32 microg/g. Higher levels were determined by dilution to fit the standard curve. Through the SLV, the method was optimized for quantification by liquid chromatography with ultraviolet detection (LC-UV) and LC/mass spectrometry (MS) confirmation. The test samples were extracted with organic solvent and water, then injected on a reverse phase LC column. Quantification was achieved with linear regression using a laboratory automation system. The SLV study included systematically optimizing the LC-UV method with regard to test sample size, fine grinding of solids, and solvent extraction efficiency. These parameters were varied in increments (and in separate optimization studies), in order to ensure that each parameter was individually studied; the test results include corresponding tables of parameter variations. In addition, the chromatographic conditions were optimized with respect to injection volume and detection wavelength. Precision studies produced overall relative standard deviation values from 2.44 up to 8.26% for aristolochic acid I. Mean recoveries were between 100 and 103% at the 2 microg/g level, between 102 and 103% at the 10 microg/g level, and 104% at the 30 microg/g level.     

Determination of niacin in food materials by liquid chromatography using isotope dilution mass spectrometry

The availability of deuterium-labeled nicotinic acid makes stable isotope dilution mass spectromerty (MS) coupled with liquid chromatography (LC) an attractive option for the determination of the water-soluble B-vitamin niacin in food samples. A method was developed based on acid digestion, solid-phase extraction with a strong cation exchange column, and reversed-phase chromatography with a C18 column. Detection is by positive ion electrospray MS. Analysis in selected ion recording mode is subject to interference problems similar to those found with other LC determinations of niacin, but the additional selectivity of multiple reaction monitoring mode largely eliminates interference problems. The method was applied to 6 different food matrixes and to appropriate reference materials, including milk samples with niacin levels near 1 ppm. The method exhibited good accuracy, based on levels obtained for the reference materials, and relative standard deviations in the range of 0.5-5%.     

Gradient reversed-phase high-performance liquid chromatographic separation of naturally occurring retinoids

A gradient reversed-phase high-performance liquid chromatographic technique is described for the facile separation and quantitation of the naturally occurring retinoids: retinoic acid, retinol, and retinyl esters. An octadecylsilane column (Waters mu Bondapak C18) is used, with gradient elution from methanol--water (80:20) (solvent A) to 70% or 100% methanol--tetrahydrofuran (50:50) (solvent B) at 2.0 ml/min; detection is by absorbance at 325 nm. Analysis can be completed, with return to starting conditions, in 25-30 min. The method is inherently flexible: retinyl esters can be eluted as a group, with little resolution, by gradient to 100% solvent B, or mostly resolved by gradient to 70% solvent B; separation of retinoids more polar than retinoic acid can be achieved by use of greater proportions of water in solvent A. The separation of vitamin A compounds from extracts of human, rat, and pig liver and from rat kidney by this technique is described.     

Solid‐phase extraction assisted dispersive liquid–liquid microextraction based on solidification of floating organic droplet to determine sildenafil and its analogues in dietary supplements

A novel analytical method for the simultaneous determination of the concentration of sildenafil and its five analogues in dietary supplements using solid‐phase extraction assisted reversed‐phase dispersive liquid–liquid microextraction based on solidification of floating organic droplet combined with ion‐pairing liquid chromatography with an ultraviolet detector was developed. Parameters that affect extraction efficiency were systematically investigated, including the type of solid‐phase extraction cartridge, pH of the extraction environment, and the type and volume of extraction and dispersive solvent. The method linearity was in the range of 5.0–100 ng/mL for sildenafil, homosildenafil, udenafil, benzylsildenafil, and thiosildenafil and 10–100 ng/mL for acetildenafil. The coefficients of determination were ≥0.996 for all regression curves. The sensitivity values expressed as limit of detection were between 2.5 and 7.5 ng/mL. Furthermore, intraday and interday precisions expressed as relative standard deviations were less than 5.7 and 9.9%, respectively. The proposed method was successfully applied to the analysis of sildenafil and its five analogues in complex dietary supplements.     

Development and validation of a liquid chromatography method for the simultaneous determination of alpha-tocopherol, retinol and retinyl esters in human serum using a monolithic column for the monitoring of anticancer therapy side effects

Among other side effects, administration of anticancer agents is accompanied by manifestations of gastrointestinal toxicity and disturbances of antioxidant balance. The monitoring of these toxic effects in clinical practice is impeded by a dearth of reliable laboratory methods. Therefore, a simple and rapid reversed-phase high-performance liquid chromatography procedure for selective and sensitive determination of retinol, a-tocopherol, and retinyl esters (retinyl-palmitate and retinyl-stearate) in blood serum has been developed and presented in this study. A Series 200 LC HPLC instrument from Perkin Elmer (Norwalk, USA) with diode-array detector (DAD) was used for the analysis. Separations of retinol, alpha-tocopherol, retinyl-palmitate, and retinyl-stearate were performed using a Chromolith Performance RP-18e, 100 x 4.6 mm monolithic column from Merck (Darmstadt, Germany). Gradient elution was used at a flow rate of 3 mL/min; the mobile phase was methanol-water (95:5, v/v) for 0-2.1 min and methanol-2-propanol (60:40, v/v) for 2.1-4.9 min. The total time of analysis was 6 min. The injection volume was 20 microL and the analysis was performed at ambient temperature. Detection of retinol, alpha-tocopherol, and retinyl esters was carried out at 325, 295, and 330 nm, respectively. For practical assessment of the method, the vitamin A absorption test was performed on seven healthy controls as well as on six patients with non-small cell lung carcinoma or head and neck carcinoma previously treated by chemotherapy and/or radiotherapy, six patients with rectal carcinoma before chemoradiotherapy, four patients with gastrointestinal stromal tumor (GIST) before treatment with imatinib, and a breast cancer patient with chemotherapy-induced diarrhea. Present data demonstrate the feasibility of large scale HPLC determination of vitamin E, vitamin A, and retinyl esters in human serum using a silica monolithic column, and this method may represent a valuable aid in the laboratory monitoring of the toxicity of anticancer therapy.     

Simultaneous determination of water-soluble vitamins in SRM 1849 Infant/Adult Nutritional Formula powder by liquid chromatography–isotope dilution mass spectrometry

Assessing dietary intake of vitamins from all sources, including foods, dietary supplements, and fortified foods, would be aided considerably by having analytical methodologies that are capable of simultaneous determination of several vitamins. Vitamins naturally present in foods may occur in different chemical forms, with levels ranging over several orders of magnitude. Vitamins in dietary supplements and fortified foods, however, are typically added in a single chemical form, and matrix issues are usually not as complex. These sources should thus be relatively amenable to approaches that aim for simultaneous determination of multiple vitamins. Our recent work has focused on development of liquid chromatography (LC)–UV/fluorescence and LC–tandem mass spectrometry methods for the simultaneous determination of water-soluble vitamins (thiamine, niacin, pyridoxine, pantothenic acid, folic acid, biotin, and riboflavin) in dietary supplement tablets and fortified foods, such as formula powders and breakfast cereals. As part of the validation of our methods and collaboration in characterization of a new NIST SRM 1849 Infant/Adult Nutritional Formula powder, we report data on SRM 1849 using isotope dilution mass spectrometric methods. Use of available NIST Standard Reference Materials® as test matrices in our method development and validation gives a benchmark for future application of these methods. We compare three chromatographic approaches and provide data on stability of vitamin standard solutions for LC-based multiple vitamin determinations.

Determination of catecholamines and indoleamines in human urine based on intramolecular excimer-forming derivatization and fluorescence detection.

A liquid chromatographic (LC) determination of catecholamines and indoleamines is described. This is based on intramolecular excimer-forming fluorescence derivatization with 4-(1-pyrene)butanoyl chloride, followed by reversed-phase LC. The analytes, containing an amino moiety and phenolic hydroxyl moieties in a molecule, were converted to the corresponding polypyrene-labeled derivatives by one-step derivatization. They afforded intramolecular excimer fluorescence, which can clearly be discriminated from the normal fluorescence emitted from reagent blanks. The detection limits (S/N = 3) for catecholamines and indoleamines were femto-mole levels per 20-microL injection. Furthermore, this method was applied to a urine assay.