US3689221A - Fluorometric assay of primary amines - Google Patents

Fluorometric assay of primary amines Download PDF

Info

Publication number
US3689221A
US3689221A US82596A US3689221DA US3689221A US 3689221 A US3689221 A US 3689221A US 82596 A US82596 A US 82596A US 3689221D A US3689221D A US 3689221DA US 3689221 A US3689221 A US 3689221A
Authority
US
United States
Prior art keywords
ninhydrin
fluorescence
primary
assay
aryl alkyl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
US82596A
Other languages
English (en)
Inventor
Sidney Udenfriend
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
F Hoffmann La Roche AG
Hoffmann La Roche Inc
Original Assignee
F Hoffmann La Roche AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by F Hoffmann La Roche AG filed Critical F Hoffmann La Roche AG
Application granted granted Critical
Publication of US3689221A publication Critical patent/US3689221A/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material

Definitions

  • a highly sensitive assay technique for primary amine containing compounds utilizes fluorescence produced by the interaction of the primary amine compound with ninhydrin and an aryl alkyl aldehyde.
  • a preferred aryl alkyl aldehyde for the assay technique is phenylacetaldehyde.
  • the assay is useful for the qualitative and quantitative determination of amino acids, peptides, proteins and any other organic compounds containing a primary amino moiety.
  • ninhydrin as a colorimetric reagent for the detection and assay of amino acids, amines and peptides has been known in the art for nearly sixty years. More recently, the ninhydrin color reaction has found extensive application in conjunction with the newly developed amino acid analyzers for the automated microanalysis of amino acid compositions of protein hydrolysates. By the use of micro techniques, it has been possible to apply the colorimetric ninhydrin procedure at the nanomole level. However, this figure represents the limit of sensitivity of the colorimetric technique.
  • ninhydrin can yield highly fluorescent products with amine-containing compounds.
  • McCaman and Robins in their paper in J. Lab. Clin. Med. 59, 885 (1962) disclosed a fluorometric assay technique that utilizes ninhydrin in the determination of serum phenylalanine. Close has reported on a study related to the fluorescence obtained by incubating amino acids or amines with ninhydrin and n-butyraldehyde (see fluorescence assay in Biology in Medicine by S. Udenfriend, Academic Press, New York (1969) at p. 196).
  • the present invention relates to an improved assay method for the determination of organic compounds containing a primary amino group utilizing fluorescence techniques.
  • the invention is concerned with a process which involves reacting an organic compound containing a primary amino group with a certain class of aldehydes and ninhydrin to quantitatively produce a highly fluorescent material which can be detected by standard fluorometric techniques and can yield both qualitative and quantitative data relating to said primary amine.
  • An important feature of this invention is that it makes possible the detection of much smaller quantities of amines, amino acids, and peptides than have heretofore been possible utilizing standard colorimetric and fluorometric techniques.
  • a major aspect of this invention is the utility for fluorometric assays in solution.
  • a mixture of the primary amine-containing compound, the aldehyde and ninhydrin in an inert solvent containing a pH buffer is heated and the fluorescence is then measured by standard fluorometric techniques.
  • aryl alkyl aldehydes give complexes that are very highly fluorescent.
  • the aryl alkyl aldehydes comprehended by this invention are defined by the formula Ar(CH CHO 3,689,221 Patented Sept. 5, 1972 ice wherein Ar is an aromatic residue and n is an integer having a value of from 1 to 8.
  • n has a value of from 1 to 3 are particularly useful.
  • Preferred aryl alkyl aldehydes are those where n is 1 and the aromatic residue is phenyl or alkyl substituted phenyl, e.g. phenylacetaldehyde or 4-methyl phenylacetaldehyde. Phenylacetaldehyde is especially preferred.
  • the fluorescence produced by using phenylacetaldehyde as compared with that produced by using n-butyraldehyde, a reagent used in previous fluorometric assays is at least 3 to -fold greater depending upon the pH.
  • primary amine-containing compounds can beassayed by the present technique.
  • Such classes of compounds include primary amines, e.g. ethylamine, ethanolamine, tryptamine, dopamine, norepinephrine, etc.; atamino acids containing a primary amino group, e.g. glycine, alanine, leucine, valine, etc. and peptides containing a primary amino group e.g. insulin, angiotensin, leucylalanine, etc.
  • the proportions of the reactants used are not broadly critical but normally it is preferred to use a large excess of the aldehyde and ninhydrin with respect to the amine being determined so that the fluorescent product is quantitatively formed. It is generally preferred to use an excess of ninhydrin as compared with aryl alkyl aldehyde. A ten-fold excess is conveniently utilized.
  • the incubation temperature is also not critical and can be carried out over a range of from about C. to about the boiling point of the reaction medium. It is preferred, however, to utilize a temperature range of from about C. to about C.
  • the time involved in the incubation varies extensively with the substrate and the pH but is generally in the range of from about 5 minutes to about 4 hours.
  • the pH of the incubation medium is normally between about 4 and 9. However, a pH range of from about 6 to 8 is especially preferable.
  • the pH is controlled by the addition of a buffer to the reaction medium.
  • Suitable buffers are phosphate buffers and others known in the art.
  • the choice of exact pH for any particular assay is dependent upon many factors such as the substrate and the type of assay being performed. For example, at pH values around 6.0 one observes a higher fluorescence for amines and peptides than for neutral amino acids. Although, in general, the absolute intensity of the fluorescence is slightly lower at the lower pH, it offers considerable specificity for the determination of amines and peptides. As one increases the pH, going for example, from pH 6 to pH 8, the fluorescence values of amino acids are increased relative to that of amines and peptides. Also, as the pH is increased the incubation time can be decreased.
  • the limits of sensitivity for the assay procedure of the present invention can be determined for a variety of substrates and compared With those for the standard colorimetric ninhydrin assay procedure of Moore and Stein, J. Biol. Chem. 176, 367 (1948).
  • the smallest amount which can be determined by the present method is in the range of 10- to 10- moles; whereas for the colorimetric method l0 moles was the smallest amount detectable. Therefore, the fluorescent ninhydrin aldehyde method of the present invention is at least 10 to times more sensitive than the ninhydrin color reaction.
  • Fluorometric detection has an inherent advantage over colorimetric detection in that in the former method an absolute quantity is measured, whereas in the latter method 21 diflFerence between two measured values is determined, thus reducing the accuracy and limiting the minimum amount detectable.
  • the limiting factor for a fluorometric assay is the sensitivity of the detection system, i.e. the instrumentation.
  • the assay procedure of the present invention has been automated to continuously detect, qualitatively and quantitatively, primary amine-containing compounds in a sample stream. It is particularly useful to employ, as a sample stream, the efiiuent from a chromatographic system, for example, a chromatographic column for the separation of amino acids.
  • the sample stream is introduced into the automated analyzer at a constant rate and streams of reagents (ninhydrin, aryl alkyl aldehyde and buffer) are added thereto.
  • a suitable means for controlling the addition of the reagents and the flow rate of the sample stream is by use of a proportionating pump.
  • the reagents are added to the sample stream, they are thoroughly mixed with the sample stream by, for example, passing through a mixing coil.
  • the mixture is then heated at the appropriate temperature for a predetermined length of time to produce fluorescent substances. The heating is conveniently done by passing the mixture through a coil of fixed length, inserted in a constanttemperature heating bath, so that the residence time in the heated coil is known.
  • the solution after leaving the heating coil, is cooled and passed into a continuous fiow cell of a fiuorophotometer where it is fluoresced and the fluorescence continuously detected.
  • the detector of the fluorophotometer may be linked to a suitable recording, data display or print-out device.
  • Another use for the assay procedure of the present invention is for the qualitative and quantitative determination of primary amine-containing compounds in a mixture 'by a technique involving automated, successive sampling of a multiplicity of samples.
  • a fixed volume of a sample solution is introduced by utilizing, for example, a probe which dips into the sample solution.
  • the sample stream thus introduced is processed as for the automated analyzer described above.
  • the sample solutions are separated from one another within the flowing stream by methods generally used, for example, solvent or gas bubbles. If the bore of the tubing carrying the flowing stream is sufiiciently small, no additional means for separating the sample solutions from one another is necessary, since capillary action will prevent intermixing.
  • the automated presentation of sample solutions for introduction into the analyzer can be accomplished by techniques generally used in the art.
  • the automated assay procedure of the present invention can also be used in closed-loop process control systems to monitor the level of primary amine-containing compounds in a reaction medium and automatically control the reaction process by methods such as that described by Hana et al. 'in U.S. Pat. 3,306,096.
  • Another major use for the present invention is for the detection of primary amine-containing compounds on paper or thin layer chromatograms.
  • the sample is applied to the paper or thin layer plate and developed in the normal manner.
  • the paper or plate is then sprayed with a solution containing ninhydrin, the aryl alkyl aldehyde and the pH buffer as described above for the solution assays.
  • the paper or plate is then heated in high yields.
  • the choice of solvent, pH, temperature and time is controlled by the same considerations as discussed above for the solution assays.
  • the techniques used for paper and thin layer chromatograms prior to the detection procedure are those normally employed in the art.
  • the sensitivity of this method is at least 10 to times greater than commonly used colorimetric methods and 3 to 30 times greater than fluorometric methods for amine assay.
  • this method has a wide scope and has been applied to a large variety of primary amines, amino acids and peptides.
  • a third advantage of the present method is that it is readily suited to automation since the fluorescence is proportional to the concentration of amine substrate over a wide range of concentration.
  • the present technique achieves maximum sensitivity as it starts with a reagent which is not fluorescent but which yields fluorescence only on interaction with primary amine-containing compounds.
  • ammonia does not react in the present process is of great importance since ammonia is usually present in substantial amounts in protein hydrolysates and it is known that traces of ammonia in the atmosphere can limit the usefulness of known colorimetric and fluorometric methods for amine detection.
  • Another advantage of this method is that at low pH values, particularly about pH 6, far more fluorescence is obtained with peptides than with amino acids. Peptides, particularly large peptides which yield little color with the classical ninhydrin procedures can be identified by the present method. Comparison of proteins by fingerprinting methods usually requires several milligrams of protein to detect all the peptides. By utilizing the procedure of the present invention one can accomplish the same results using only microgram quantities of the protein. Automated fluorometric assays of peptides obtained from digestion of microgram quantities of proteins make it possible to carry out genetic studies even on the small amounts of protein obtained from a single laboratory animal.
  • Another important application is in the detection of small amounts of physiologically active peptides in tissues.
  • the method of the present invention is suitable for the detection of new peptides whose biological roles or activities have not yet been ascertained.
  • EXAMPLE 1 General procedure for fluorometric assay in solution To a 13 x 100 mm. test tube were added 2.0 ml. of 0.2 M phosphate bufl'er (pH 6, 7, or 8) 0.2 ml. of aqueous ninhydrin (50 mM.), 0.1 ml. of aqueous sample solution (generally 10* to 10- mM.) and finally 0.1 ml. of phenylacetaldehyde in ethanol (10 mM.). After mixing thoroughly, the tubes were covered with aluminum foil and transferred to a 60 water bath for a suitable incubation period; 15 min. at pH 8.0, 60 min. at pH 7.0 and min. at pH 6.0.
  • Example 1 The general procedure of Example 1 was used, employ- 3 ing 10 III/1.1110165 of each amino acid and 120 minutes Twenty ,ul. of each aldehyde in ethanol solution (10 mM.) was added to a mixture containing 40 ,ul. of leucylalaninglycylvaline (1.25 mM.), 75 1.1. of ninhydrin (30 mM.) and 200 l. of 0.6 M phosphate bufler at the pH indicated. After incubating for 90 minutes at 60 the samples were cooled to room temperature and fluorescence was measured as described in Example 1.
  • EXAMPLE 7 This example illustrates the relative fluorescence intensity of some biologically active peptides and amines.
  • EXAMPLE 8 This example illustrates the difference in sensitivity between the fluorescence assay of the present invention and the standard colorimetric assay.
  • EXAMPLE 9 Varying amounts of glutamic acid, glycine, leucylalanine and tyramine in 1 ,ul. were spotted on paper or silica gel thin layer plates. After development with solvents and drying, the paper or plate was sprayed with a solution of phenylacetaldehyde (15 mM.) and ninhydrin mM.) in ethanol. The ethanol was allowed to evaporate at room temperature and the chromatogram was then sprayed with 0.2 M phosphate buffer, pH 8.0.
  • the detection limit for glutamic acid was 10" mole; for glycine, leucylalanine and tyramine, 10- mole. With the normal Ninhydrin color reaction, the detection limit was 10- mole for all four.
  • EXAMPLE 10 Ten #1. of an amino acid standard solution (containing l mole/ml each of common amino acids and ammonia) was applied to a 0.9 cm. x 55 cm. column of 7.5% cross-linked sulfonated styrene copolymer resin (Beckman UR- resin for the separation of acidic and neutral amino acids) and developed at 70 ml./hr., first with 117 ml. 0.2 N sodium citrate, pH 3.25, then with 140 ml. 0.2 N sodium citrate, pH 4.3. The starting temperature was and after 30 minutes it was raised over a 1 hour-period to and held at this temperature throughout the remainder of the experiment.
  • an amino acid standard solution containing l mole/ml each of common amino acids and ammonia
  • the efiluent from the column was split into two streams in the ratio of 9: 1.
  • the larger of the streams was passed through a Beckman Model 120-'C amino acid Analyzer in the normal fashion.
  • the smaller stream, flowing at 0.1 ml./min. was mixed with 0.3 mL/min. of a stream of a solution prepared by mixing 2.67 g. ninhydrin, 180 mg. phenylacetaldehyde, 300 ml. of ethanol and 150 ml. water, and a stream of 0.2 M sodium phosphate buffer, pH 7.5, flowing at 2.0 mL/min.
  • These flow rates were controlled by means of a proportionating pump.
  • the total flow rate after mixture was 2.4 ml./min.
  • the fluorophotometer was operated at a high voltage setting of 40, a sensitivity setting of 50% and an attenuation setting of 3% full scale (corresponding to 3 mv. for full scale reading).
  • the output of the fiuorophotometer was coupled to a Beckman 10 inch recorder with a chart speed of 0.1 inch/min.
  • the recorder used in the Beckman Amino Acid Analyzer for the normal colorimetric analysis was operated at a 5 mv. full scale sensitivity and recorded the normal 570 m output of the colorimeter.
  • the chart speed was 0.1 inch/min.
  • the attached drawing shows the recorder tracings made under the above-described conditions.
  • the first run (shown at the left) is for the basic amino acids using the PA-35 column.
  • the second run is for the neutral and acidic amino acids using the UR-3O column.
  • the chart below presents the data of the attached drawing in tabular form.
  • the relative areas under each peak recorded by both the colorimetric and fiuorometric methods is presented. These areas have been corrected for differences in the amount of sample being measured by each method and difference .in the attenuation of the recorders used.
  • the figures as presented represent the integrated response of each detector under identical conditions produced by equal amounts of each amino acid.
  • a method for determining a primary amine-containing compound in a mixture containing same which method comprises contacting said mixture with ninhydrin and an aryl alkyl aldehyde selected from the group consisting of phenylacetaldehyde and 4-methylphenylacetaldehyde heating the above treated mixture to produce a fluorescent substance therein fluorescing said substance and detecting the fluorescence.
  • concentration in solution of a known primary amine-containing compound is quantitatively determined by (a) determining the fluorescence produced by a measured concentration of said amine, (b) determining the fluorescence produced by the unknown concentration of said amine, and (c) obtaining the unknown concentration by multiplying the known concentration by the ratio of the unknown to the known fluorescence values.
  • the primary aminecontaining compound is determined by an automated procedure which comprises (a) continuously contacting a sample stream containing said primary amine-containing compound in solution with a fixed proportion of ninhydrin and aryl alkyl aldehyde, (b) continuously passing the mixture obtained in step (a) through a heating zone to produce a stream containing a fluorescent substance, (c) passing the solution obtained in step (b) at constant rate through a flow cell of a fluorophotometer whereby any fluorescent material present is fiuoresced and detected.
  • sample stream is at least a portion of the eflluent of a chromatographic column.
  • a reagent for the determination of primary aminecontaining compounds consisting essentially of a solution of ninhydrin and an aryl alkyl aldehyde selected from the group consisting of phenylacetaldehyde and 4-methylphenylacetaldehyde in an inert organic solvent.

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Optics & Photonics (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
US82596A 1970-10-21 1970-10-21 Fluorometric assay of primary amines Expired - Lifetime US3689221A (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
US8259670A 1970-10-21 1970-10-21

Publications (1)

Publication Number Publication Date
US3689221A true US3689221A (en) 1972-09-05

Family

ID=22172164

Family Applications (1)

Application Number Title Priority Date Filing Date
US82596A Expired - Lifetime US3689221A (en) 1970-10-21 1970-10-21 Fluorometric assay of primary amines

Country Status (9)

Country Link
US (1) US3689221A (enrdf_load_stackoverflow)
AU (1) AU3442271A (enrdf_load_stackoverflow)
DD (1) DD94907A5 (enrdf_load_stackoverflow)
DE (1) DE2148603A1 (enrdf_load_stackoverflow)
FR (1) FR2110033A5 (enrdf_load_stackoverflow)
GB (1) GB1349491A (enrdf_load_stackoverflow)
IL (1) IL37847A0 (enrdf_load_stackoverflow)
NL (1) NL7114508A (enrdf_load_stackoverflow)
ZA (1) ZA716604B (enrdf_load_stackoverflow)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3830629A (en) * 1972-10-18 1974-08-20 Hoffmann La Roche Fluorometric analysis of secondary alpha-amino acids
US3871825A (en) * 1971-12-27 1975-03-18 Hoffmann La Roche Fluorometric reagents and methods
US3892530A (en) * 1974-09-23 1975-07-01 Hoffmann La Roche Colorimetric and fluorometric method
FR2309866A1 (fr) * 1975-04-30 1976-11-26 Foss Electric As N Procede et reactif pour l'analyse quantitative de la l-lysine dans des substances proteiques
US4253848A (en) * 1980-01-21 1981-03-03 The United States Of America As Represented By The Secretary Of The Army Method for determining oxidative status of unsaturated lipids and method for evaluating antioxidant effectiveness in lipid-polypeptide layers
US4274833A (en) * 1979-10-18 1981-06-23 Pickering Michael V Ninhydrin reagent for use in amine and amino acid analyses
US6696304B1 (en) 1999-02-24 2004-02-24 Luminex Corporation Particulate solid phase immobilized protein quantitation
CN110687235A (zh) * 2019-10-18 2020-01-14 贵州省烟草科学研究院 一种叔胺手性色谱分析中去除伯、仲胺干扰的衍生化与反萃取方法

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2508147B (en) * 2012-11-21 2016-06-01 Jpp Chromatography Ltd Ninhydrin reagent for use in a method of analysing nitrogen-containing compounds
GB2508149B (en) 2012-11-21 2015-12-09 Jpp Chromatography Ltd A method for analysing amino acids

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3871825A (en) * 1971-12-27 1975-03-18 Hoffmann La Roche Fluorometric reagents and methods
US3830629A (en) * 1972-10-18 1974-08-20 Hoffmann La Roche Fluorometric analysis of secondary alpha-amino acids
US3892530A (en) * 1974-09-23 1975-07-01 Hoffmann La Roche Colorimetric and fluorometric method
FR2309866A1 (fr) * 1975-04-30 1976-11-26 Foss Electric As N Procede et reactif pour l'analyse quantitative de la l-lysine dans des substances proteiques
US3994688A (en) * 1975-04-30 1976-11-30 A/S N. Foss Electric Method and reagent for quantitative analysis of l-lysine in proteinaceous test substances
US4274833A (en) * 1979-10-18 1981-06-23 Pickering Michael V Ninhydrin reagent for use in amine and amino acid analyses
US4253848A (en) * 1980-01-21 1981-03-03 The United States Of America As Represented By The Secretary Of The Army Method for determining oxidative status of unsaturated lipids and method for evaluating antioxidant effectiveness in lipid-polypeptide layers
US6696304B1 (en) 1999-02-24 2004-02-24 Luminex Corporation Particulate solid phase immobilized protein quantitation
CN110687235A (zh) * 2019-10-18 2020-01-14 贵州省烟草科学研究院 一种叔胺手性色谱分析中去除伯、仲胺干扰的衍生化与反萃取方法

Also Published As

Publication number Publication date
DD94907A5 (enrdf_load_stackoverflow) 1973-01-05
GB1349491A (en) 1974-04-03
DE2148603A1 (de) 1972-05-04
NL7114508A (enrdf_load_stackoverflow) 1972-04-25
FR2110033A5 (enrdf_load_stackoverflow) 1972-05-26
ZA716604B (en) 1972-06-28
IL37847A0 (en) 1971-12-29
AU3442271A (en) 1973-04-19

Similar Documents

Publication Publication Date Title
Morse et al. Thin-layer chromatographic separation of DNS-amino acids
Samejima et al. Condensation of ninhydrin with aldehydes and primary amines to yield highly fluorescent ternary products: II. Application to the Detection and Assay of Peptides, Amino Acids, Amines, and Amino Sugars
US4257774A (en) Intercalation inhibition assay for compounds that interact with DNA or RNA
Lai [26] Detection of peptides by fluorescence methods
US3689221A (en) Fluorometric assay of primary amines
WO1994017409A1 (en) Quantifying trace analytes by affinity capillary electrophoresis
CA2121842A1 (en) Non-radioactive enzyme assay
EP0141298A1 (en) Analytical method and element for albumin
Roth Fluorimetric determination of free hydroxyproline and proline in blood plasma
Zhu et al. Post-column derivatization for fluorescence and chemiluminescence detection in capillary electrophoresis
BR112013007614B1 (pt) método para medir um analito, composição reagente, mistura reagente, uso de uma composição reagente, uso de uma amida de ácido carbônico e kit
Josefsson et al. An automated fluorescence method for the determination of total amino acids in natural waters
Randall et al. Approaches to the analysis of peptides
US20100190658A1 (en) Colorimetric assay for the visual detection of primary and secondary amines
De Silva et al. Direct detection of inorganic ions and underivatized amino acids in seconds using high-speed capillary electrophoresis coupled with back-scatter interferometry
Švedas et al. The interaction of amino acids with o-phthaldialdehyde: a kinetic study and spectrophotometric assay of the reaction product
US3817706A (en) Fluorescence quantitative thin layer chromatographic method
Bisker et al. A new sensitive method for the quantitative evaluation of the hydroxyproline isomers
CA2315051C (en) Capillary electrophoretic methods to detect new biologically active compounds in complex biological material
US5252492A (en) Fluorescence assay for ligand or receptor utilizing size exclusion
US20080199967A1 (en) System for Detection of S-Nitrosoproteins
US3830629A (en) Fluorometric analysis of secondary alpha-amino acids
Shore et al. Quantitative determination of amino acids by fluorescence of derivatives on paper
Kai et al. Fluorescence derivatization of bioactive peptides for high-performance liquid chromatography
Boppana et al. High-performance liquid chromatographic determination of peptide drugs in biological fluids by means of pre-and post-column fluorescence derivatization techniques