J. clin. Path., 1970, 23, 315-318

Thin-layer chromatography of trinitrophenylderivatives of amino acids in urine and plasma

D. PHILPOTTFrom the Department ofPathology, General Hospital, Northampton

SYNOPSIS The preparation and separation by thin-layer chromatography of the trinitro-phenyl derivatives of amino acids in plasma and urine is described. The semi-quantitativeprocedure presents a useful addition to existing methods for screening biological fluids foraminoacidopathies.

The use of 2,4,6-trinitrobenzene-1-sulphonicacid (TNBS) for the preparation of the trinitro-phenyl derivatives of amino acids and peptideswas described by Okuyama and Satake (1960)and Satake, Okuyama, Ohashi, and Shinoda(1960). It was observed by Nitecki, Stoltenberg,and Goodman (1967) that mixtures of trinitro-phenyl-amino acids could be resolved remarkablywell by thin-layer chromatography. They furthernoted that the preparation of these derivativespresented a number of distinct advantages overthat of dinitrophenyl amino acid derivativesprepared with the aid of 1-fluoro-2,4-dinitro-benzene. The TNBS reagent is water soluble,resulting in homogeneous reaction mixtures,and side reactions are largely avoided.The technique of Nitecki et al (1967) has been

applied here, with some modification, to urineand plasma, the amino acid profiles being in-vestigated densitometrically.

Materials

Analytical or 'special for chromatography'grade reagents were employed when available.The chloroform used contained 2% v/v ofethanol.

Thin-layer chromatography plates, 5 x 20 cm,and cylindrical jars, 6 5 cm diameter, approxi-mately 22 cm high, and with ground glass lids,

were supplied by Camlab (Glass) Ltd, Cambridge.Silica gel G1 (Kiesel G nach Stahl) was used.

Distilled water, was added to 30g silica gel ina 500-ml conical flask fitted with a rubber bung.The flask was shaken vigorously by hand for2 min before pouring the contents immediatelyinto a Quickfit & Quartz spreader. This quantityof slurry was sufficient to coat 20 plates with alayer 0-25 mm thick. The plates were airdried for 24 hours before use. No activationwas employed.

Methods

As changes occur in urine and plasma con-stituents in a few hours at room temperature,all specimens were stored at -20°C until readyfor processing.

It should be established that the patientunder investigation is not receiving drugs,of which metabolites in the urine would formTNP derivatives. Examples are penicillaminein the urine of patients receiving penicillin,and nitrazepam (Mogadon), which is partlyexcreted in a reduced form, the nitro groupbeing converted to an amino group.1Anderman & Co Ltd, Battlebridge House, Tooley Street,

London.

Received for publication 11 September 1969.

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316 D. Philpott

1 NEUTRAL AND ACIDIC AMINO ACIDDERIVATIVES

UrineTo 1 ml of specimen in a glass-stoppered centri-fuge tube was added 1 ml of water, 1 ml of 4%sodium bicarbonate solution, and a knife-point(50 mg approximately) of 2,4,6-trinitrobenzene-I-sulphuric acid. In the case of very dilutespecimens, 2 ml undiluted urine was taken.The mixture was kept for two hours in the darkat 37°C, cooled, and then acidified with 1 ml1N hydrochloric acid. The trinitrophenyl aminoacid derivatives were extracted into two 5 mlportions of ether, and centrifuged briefly toassist separation. The combined extracts weredried over anhydrous sodium sulphate and thenevaporated to dryness in a water bath. The residuewas cooled and dissolved in 0-1 ml acetone,transferred to a small tube, and stored, wellstoppered, in the dark at -20°C.

PlasmaProtein was removed by heat coagulation.To 1 ml of plasma in a small test tube was added1 ml of 0-05N acetic acid. The mixture was heatedin a boiling water bath for 3 min, cooled, andcentrifuged. The supernatant fluid was decaniedinto a glass-stoppered centrifuge tube, subsequentprocessing being as described for urine.

2 BASIC AMINO ACID DERIVATIVES(URINE AND PLASMA)Any residual ether remaining after extractionwas pipetted off. Acetone, 0 5 ml, was addedto the residual fluid and the mixture was saturatedwith solid ammonium sulphate. Aftercentrifuging,the acetone layer was quantitatively transferredto a small tube and stored at -20°C.

CHROMATOGRAPHY

Aliquots of the acetone solutions derived from 1(10 [l of the urine extract and 20 pl of the plasmaextract) were applied with micropipettes at points2-5 cm from the end of a 5 x 20 cm silica gel-coated plate to provide spots not exceeding 4 mmdiameter and developed with 30 ml of a freshlyprepared 9:1 v/v mixture of chloroform andglacial acetic acid in a cylindrical jar with aground-glass lid. The solvent front was allowedto advance to a horizontal line marked on theplate 10 cm distant from the point of application.This required approximately 35 minutes. Thedeveloped plate was allowed to dry in the draughtof a fume cupboard for 10 minutes.

Portions, 20 ,ul, of the acetone solutions,derived from 2 (plasma or urine), were similarlychromatographed in a freshly prepared solventmixture composed of 30 ml n-butanol and 1 ml

1N hydrochloride acid. This took approximately120 minutes.Whereas all trinitrophenyl amino acid de-

rivatives were visible initially as yellow spots onthe plate, darkening was observed in varyingdegree on exposure to light and air, particularlyin the case of the sulphur-containing aminoacids. The procedures were therefore executedthroughout in subdued light and densitometrywas undertaken without delay after drying theplates.

DENSITOMETRYRecording of the developed plates was carriedout with a Vitatron densitometer employingan OBI Chance blue filter and 3 x 0-2 mm slit.A pinpoint size hole made in the gel at the pointof application of the extract was recorded inthe tracing as a narrow rectangular trough,as was also the line marking the solvent front,thus enabling exact measurement of Rr values.When one or more derivatives were found presentin excess amounts more accurate values wereobtained by repeating the chromatogram withsmaller aliquots of the acetone solution.

Results

The excellent separation of mixtures of trinitro-phenyl derivatives of amino acids obtained byNitecki et al (1967) by thin-layer chromatographywith pure solutions was repeated when appliedto urine and plasma. Nine to 10 clearly definedspots of trinitrophenyl derivatives of neutraland acidic amino acids were normally obtainedand were recorded as well defined peaks onthe densitometer tracings. They were invariablypreceded on the plate in both urine and plasmachromatograms by the crescent-shaped spotof picramide. This showed intense yellowfluorescence under ultraviolet light at 350 m,u.Its presence has been attributed to the action ofammonia, derived from glutamine, on the TNBSreagent. Urea, too, was found here to be 'hydro-lysed' to some extent to produce picramide insimilar fashion. Fortuitously, the picramide spotformed a useful marker to which the trinitro-phenyl amino acid derivatives could be referred.They have been designated Rp values andwere adopted in preference to Rf values dueto their greater reproducibility. With care in thepreparation of the plates (Rp x 100) values(Table I) were found to be reproducible within± 2. Typical chromatograms of trinitrophenylderivatives of neutral and acidic amino acidsderived from urine and plasma are illustrated inFigures 1-3. The small amount of picric acidproduced and extracted remained at or near theorigin.Chromatography of the ether-insoluble basic

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Thin-layer chromatography of trinitrophenyl derivatives ofamino acids in urine andplasma

U)I- < (D (.D

~ 1

< a.

Fig. 1 Urine, adult, showing Pic, GNH2, Ser, Thr,Asp, Glu, Gly, Try, Ala, Ph.Al, Leu, Picr.

, =D >, >

U) F < 0 0

A I .t

Amino Acid

CitrullineGlutamineCystineAsparagineCystathionineSerineThreonineAspartic acidHomocystineGlutamic acidTyrosineCysteineGlycineHomocysteineOrnithineTryptophanAlanineLysineMethioninePhenylalanineP-Alaninea-Aminobutyric acidP-Aminoisobutyric acidLeucineIsoleucineValine

KynurenineCarnosinePenicillanine

Fig. 2 Plasma (child, aged 2 yr, epileptic), showingPic, GNH2, Ser, Thr, Asp, Glu, Gly, Try, Ala,Ph.AI, a-NH2Bu, Leu, Picr.

Amino Acid Rf x 100

Taurine 6Ornithine 10 (broad), 83Lysine 10 (broad), 863-Methyl-histidine 16I-Methyl-histidine 21 (broad)Arginine 37Histidine 37 (major), 58 (minor)Cystine 62Homocystine 63

Table II Derivatives of basic amino acids

Key to Figures I and 2a-Aminobutyric acid = a-NH,Bu; alanine = Ala;aspartic acid = Asp; glutamicacid= Glu; glutamine =GNH2; glycine = Gly; histidine = Hist; 3-methyl-histidine = 3-meth-hist; homocystine = homocyst,leucine = Leu; methionine = Met; phenylalanine;Ph.Al; picric acid = Pic; picramide = Picr.

amino acid TNP derivatives presented somedifficulties. The salting-out procedure adoptedhere proved more satisfactory than the directspotting of the acid reaction mixturesonto the plates proposed by the originalauthors. The acid-butanol solvent mixturefinally adopted was the best of a wide rangeof solvent mixtures investigated. Figure 4illustrates a typical chromatogram of the trinitro-phenyl derivatives of basic amino acids in a

normal urine. Rf values are listed in Table II.

In the case of diamino acids, where mono-and bis-substitution is possible, ether-soluble

and ether-insoluble derivatives may be formed.Corresponding reference values are listed in thetables.

Quantitative determination of fluid aminoacid content by integration of the densitometerrecordings was not undertaken, as the aminoacids were found not to react uniformly withthe TNBS reagent. It was noted, however, thaton acidification of the reaction mixture beforeether extraction, fluids of normal amino acidcontent remained clear, or at most exhibiteda slight haziness. Formation of a precipitateat this stage would indicate the presence of an

IuZ

RV x 100

ICU z

(

5

89, 391014, 2516213033, 613538475262636578828286899092949497

9, 75, 858573, 89

Table I Derivatives of neutral and acidic aminoacids

(a11; 15 = IL)-1- i< CL --- 3 E

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318 D. Philpott

Fig. 3 Urine from a case of homocystinuria11-year-old boy showing Pic, GNH2, Ser, ThrGlu, Gly, Homocyst, Met, a-NH2Bu, Picr.

aminoaciduria, unless a specimen of urine ofhigh specific gravity was involved. Semiquanti-tive assessment for screening purposes wasfurther provided by comparison of the densito-meter tracings with those obtained from normalfluids. Normally in urine the heights of the peaksrarely exceeded that given by the picramidespot.

Conclusions

The technique described has the merit for screen-ing purposes of speed, simplicity, and, for themost part, excellent chromatographic resolution.The use of heat coagulation for plasma proteinprecipitation has the advantage of being rapidbut is likely to produce artefacts. However,comparison of the results with those obtainedusing the solvent extraction technique of Naftalinand Stephens (1965) revealed no significantdifferences.One disadvantage of the method is the failure

of proline and hydroxyproline to form trinitroderivatives since the reagent reacts only with theprimary amino groups. Brown (1968), however,has recently recorded a derivative of proline

in a formed on prolonged reaction in a more alkaline, Asp, medium and it may prove possible to adopt the

method accordingly.

The assistance of Mr G. B. Dun, medical photographerto the hospital, is gratefully acknowledged.

Fig. 4 Urine, adult, showing basic amino acids3-meth-hist, Hist (two peaks).

Key to Figures 3 and 4a-Aminobutyric acid = a-NH2Bu; alanine = Ala;aspartic acid= Asp; glutamic acid= Glu; glutamine;GNH2; glycine = Gly; histidine = Hist; 3-methyl-histidine = 3-meth-hist; homocystine = homocyst =leucine =Leu; methionine = Met; phenylalane =Ph.Al; picric acid = Pic; picramide = Picr.

References

Brown, H. H. (1968). A study of 2,4,6-trinitrobenzene-sulfonicacid for automated amino acid chromatography. Clin.Chem., 14, 967-978.

Naftalin, L., and Stephens, A. (1965). A method for the de-termination of free plasma amino acids in ultra-microquantities. Clin. chim. Acta, 12, 365-369.

Nitecki, D. E., and Stoltenberg, 1. M., and Goodman, J. W.(1967). Qualitative and quantitative determination ofmixtures of amino acids using 2,4,6-trinitrobenzene-sulfonic acid. Analyt. Biochem., 19, 344-350.

Okuyama, T., and Satake, K. (1960). On the preparation andproperties of 2,4,6-trinitrophenyl amino acids and pep-tides. J. Biochem. (Tokyo), 47, 454-466.

Satake, K., Okuyama, T., Ohashi, M., and Shinoda, T. (1960).The spectrophotometric determination of amines, aminoacids, and peptides with 2,4,6-trinitrobenzene-l-sulfonicacid. J. Biochem. (Tokyo), 47,654-660,

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