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Phosphorescence characteristics of several antimetabolites
Tahata, 1969,Vol. 16.pp. 407to 409. PcrpamonPIWE. Printedin NorthernIreland
SHORT COMMUNICATIONS
Ph~phor~en~e characteristics of several an~e~~~t~ (Received 8 Augast 1968. Accepted 21 August 1968) PHOSPHORIMTRYhas become widely accepted as au analytical tool for the study and analysis of compounds of biochemical interest.1 It has reviously been used to determine the n&tine alkaloids in tobacco,* drugs in biological fluids,*-7 an B pesticides in foods and in biological flulds.8*e In this study, the ph~hor~~~ characteristics of 37 ~time~bolit~ were determined, and the possible application of phosphorhnetrlc techniques to the determination of 17 of these compounds in blological materials is indicated. The antimetabolltes studied are of considerable importance in plant and animalgrowth. EXPERIMENTAL
Apparatus All uhosuhorimetric measurements were taken w&h the Aminco Bowman s~~~hotoffuorometer (No. 4-21202)with the phosphorous at&&merit (No. C 27-62140) and-a xe&n lamp (No.
901 Gl; all items manufactured by American Instrument Company, Inc., Silver Spring, Maryland). All studies were made with the slit programme: A 4 mm, B 3 mm, C 3 mm, D 4 mm and E 3 mm. All ;F were recorded with an Aminco X-Y recorder (No. 1620-827, American Instrumeut Company, Reagents All ~~e~~lit~ were purchased in a growth factor analogue kit (Nutritional Biochemicals ~rporation, Cleveland, Ohio). Absolute ethanol (Union Carbule Corporation) purl5ed as pre-
viously described,” was used as the solvent for all phosphorimetrlc measurements. Stock ethanollc solutions (approximate1 lO-*jV) of each corn ound were prepared. Solutions of lower concentrations were prepared 2:y successive dilution. !I than01 was used as the solvent because it is lnexpensive, can be prepared in a highly pure state, and freexes to a clear, rigid glass at 77%. Procedures Ana&tical curves (logarithm of phosph~e~c
signal uersus logarithm of inaction) were obtained to determine whether a compound was analytically useful, and its lit of detection. The limit of detection was de5nedlQ as the concentration which gave a signal-to-noise ratio of tt/s/&. The noise was estimated to be one 5fth of the range of 5uctuation in the background signal, t was the Student t, and n was the mnnber of combined blank and sample measurements made at the limit of detection. With n equal to six and a 99 % con5dence level, the signal-to-noise ratio is approximately 2, The lifetime,r, was measuredby terminating the exciting radiation with a mamnd shutter and plotting the phosphorhnetric signal aemus time wtth the X-Y recorder. The response of the recorder prevented any r hornets shorter than O-5sec. RESULTS AND DISCUSSION Seventeen antlmetabolites having analytically useful phosphorescence are given in Table I. In addition to these 17 compounds, barbituric acid, gamma hexachloro benxene, Dr.,-~fluorophenylalanine, pyrldme-3-&phonic actd, @-2-thienylala&ne, B-2-thlenylseriue, and DL-,8-%hif@alanine phosphoresced, but too weakly to be considered analytically useful. The remaining 12 authnetabolltes didnot phosphoresce [ally1 lyclue, aminoacetic acid @ycine), r+-arglnlne hy~~~o~~, tasparagine, nr.-leucme, n&u&e, ~mnethionim L-canavauine sulphate, %esthlobiotin, ~~y~hol~te~l, sulphoxlde, Dr.-a-methylglutamic acid and sodium salt of pantoyl taurinel. The limits of detection of the antimetabolites listed in Table I compare favourably with those attained by currently accepted calorimetric (0*1-l &ml) and enxymlc (O-0141 @ml) procedures. Previous studies of conjugated rlug system analogues showed that phosphorecence could be enhanced 407
408
short commuIlications
TABLBI.-PHOSPHO RIMmmc cHARAt2lxusn
CSOFSW3RALANTIMBT
ABOLlT@S
Excitation maximum,*t
Emission maximum*t,
Limit of detection,
Lifetime:
Antimetabolite
nm
nm
Pglml
set
3-Acetylpyridine Adenosine 2-Amine-4-methylpyrimidine L-3-3Aminotyrosine dihydrochloride 8-Azaguanine Benzimidazole 2-Chloro-4aminobenzoic acid Desoxypyridoxine hydrochlonde 2,6-Diaminopurine sulphate E%radiol Oxythiamine hydrochloride IX+-Phenyl-lactic acid c+Picolinic acid hydrochloride Pyr~re+&sulphonic acid
277 280 302
:; 438
:::o 0.033
286 282 280
398 442 406
0.81 0.30 0.0059
2.4 ;:;
loas lO?j lol§
312
447
0.069
1.0
10’
290
442
0.076
1.4
lo”
294 292 272 262
424 403 460 383
278
400
0014
5.2
101
272 343 297 312
408 480 411 432
4.8 0.30 0.012 0.0038
1.2
lO?j lP§ 1058 lW§
Quercetin Sulphanilamide 2-Thiouracil
Concentration range of near linearity
CO.5
::;9 ::;
;:;
1.7 2.0 <0*5 5.4
;:;
10‘8 10’1 lO?j
lO?j 1oq IO”8 1oq
* Peaks are uncorrected for instrumental characteristics. t Relative error of f5nm in wavelength of peaks. $ Limiting value of lifetime (0.5 set) is determined by recorder response time. 0 Up r concentration (lO-*M) is still on linear portion of analytical curve. Therefore actual range wt*E”be greater than that reported. by selective substitution on the ring(s) with corresponding improvement of the liits of detection. Therefore, values for limits of detection found in Table I indicate promise and need for further work on analogues of pharmacological interest. Acknowle@exrex~-This work was carried out as part of a study on the hosphorimetric analysis of drugs in blood and urine, supported by the U.S. Public Health Service PGM-11373-05). Department of Chetitry University of Rorida Gainesville, Floriala 32601, U.S.A.
L. B. SANDBRS J. J. Cxroax~~~ J. D. WINEFORDN~R
SummarJr-Phosphorescence excitation and emission wavelength peaks, lifetimes, limits of detection, and concentration ranges of analytical usefulness of 37 antimetabolites in rigid (77’K) ethanolic solution were determined. Seventeen of the metabolites produced analytically useful phosphorescence, whereas the remaining 20 were of limited or no analytical use. ZusammeafaaPrmg-Die We&m&en der Anregungs- und Emissionsmaxima der Phosphoreszenz, Lebensdauem, Nachweisgrenzen und analytisch nutzbare Konzentrationsbereiche von 37 Antimetaboliten in glasartiger (77°K) %thanolischer Ltisung wurden ermittelt. Siebzehn der Metaboliten zeigen analytisch nutzbare Phosphoreszenz, die Ubrigen 20 waren von beschranktem oder keinem analytischen Nutzen.
Short c4mmlmications
409
R&tun&On a d&rmln6 les longueurs d’onde des maximums d’excitation et d’&nission de pho horescen ce, les d&es de vie, les limites de d6tection et les domaines 7 e concentration d’utilite analytique pour 37 antim6tabolites en solution 6thanolique rigide (77°K). Dix sept des m6tabolites produisent une phosphorescence analytiquement utile, tandis que les 20 restant sont d’emploi analytique limit6 ou nul. REFERENCES 1. J. D. Wineforclner, W. J. McCarthy, and P. A. St. John, Phospharimetry as an Analytical Approach in Biochemistry, in Metbo& of Biochemical Analysis, D. Glick, editor, Vol. 15, Interscience. New York, 1967. 2. J. D. Winefordner and H. A. Moye, Anal. Chim. Acta, 1965,32,278. 3. J. D. Winefordner and H. W. Latx, Anal. Cbem., 1963,35,1517. 4. J. D. Winefordner and M. Tin, Anal. Cbim. Acta. 1964,31,239. 5. H. C. Hollitield and J. D. Winefordner, ibid., 1966,36,351. 6. I&m, TaIanta, 1965, 12,860. 7. R. H. Stille and A. Srent-Gyorgyi, Proc. Natl. Acad. Sci. U.S.A., 1957, 43,477. 8. H. A. Move and J. D. Wmefordner. - J. Apr. Food Chem.._ 1965.13,516. . Y
9. Idem, ibid:, 1965, 13,533.
10. J. D. Winefordner, W. J. McCarthy, and P. A. St. John, Anal. Chem., 1967,39,1495.
Tdmta, 1969,Vol. 16,pp. 409to 412. permmonPress. primedin NorthernIdand
Separation of zirconium by thin-layer chromatograplty (Received 20 May 1968. Accepted 29 .Wy 1968) RBWRSD-PHASE thin-layer chromatography has been used for the separation of xirconlum,lJ but normal-phase thin-layer chromatography has scarcely been used. Galiardi and Pokomp separated Zr(IV), Ni(II), Al(m), Ti(IV), Mn(II), Cd(n), V(V), IJO, Fe0 and ZnO by development with acetone-6M hydrochloric acid-dioxan or ethyl methyl ketone on silica 1 layers. Breccia and Spalletti6 separated zirconium-95 and niobium-95 by eluting with methano K” -lOM hydrotbroric acid (25:l) on silica gel layers. Moghissi@separated these radionuclides by eluting with 025M oxalic acid and O-lit4 hydrochloric acid in ethyl methyl ketone, dioxan and water (5:l:l) on Kieselguhr or Kieselgel layers. The present work was, therefore, undertaken to provide more simple and effective TLC systems for the separation involving Zr. SC.Y. Th. U(VI), rare earth and other metal ions. Zirconium remains at the starting point on s&a gel-cellulsoe (5: 1) plates in mesityl oxide-ethanol-nitric acid media, whilst 19 of the other metal ions tested have lamer R, values. extent silver. which tails. This forms the basis of a very selective method for the +ation of zircon&i. ’
EXPERIMENTAL Reagents Test solutions of metal ions, @lM. Appropriate amounts of the nitrates of IJO?), Th, Al, Pb,
Co(B) and Ag were dissolved in 1M nitric acid. Appropriate amounts of the oxides or carbonates of Fe(III), Zn, Cd, Cu(II), Ni, Sc, Y, La, Sm, In, Bi(III), Hg(II), Mg and Ca were converted into the nitrate by treatment with excess of nitric acid, followed by evaporation to dryness. The resulting nitrates were dissolved in 1M nitric acid. Zirconyl chloride was dissolved in dilute nitrrc acid and the solution made ammoniacal. The precipitate of zirconium hydroxide was filtered off and washed free from chloride with demineralized water, and dissolved in nitric acid. The solution was evaporated to dryness and the residue dissolved in 1M nitric acid. Preparation of thin-layer plates. Silica gel (25 g, WAKOGEL B-O, Wako Pure Chemical Industries Co. Ltd., Osaka, Japan) purified according to Seiler’s method,’ and cellulose powder MN-300 (5 g, Machery , Nagel Co., Ltd., Dtlren, Germany) were mixed thoroughly with 87 ml of demineralired water. The slurry was spread 250 p thick on 100 x 100 mm or 200 x 200 mm glass plates. The thin-layer plates were dried first in air for 1 hr and then in an oven at 105-110” for 1 hr and were stored in a desiccator till used.
SHORT COMMUNICATIONS
Ph~phor~en~e characteristics of several an~e~~~t~ (Received 8 Augast 1968. Accepted 21 August 1968) PHOSPHORIMTRYhas become widely accepted as au analytical tool for the study and analysis of compounds of biochemical interest.1 It has reviously been used to determine the n&tine alkaloids in tobacco,* drugs in biological fluids,*-7 an B pesticides in foods and in biological flulds.8*e In this study, the ph~hor~~~ characteristics of 37 ~time~bolit~ were determined, and the possible application of phosphorhnetrlc techniques to the determination of 17 of these compounds in blological materials is indicated. The antimetabolltes studied are of considerable importance in plant and animalgrowth. EXPERIMENTAL
Apparatus All uhosuhorimetric measurements were taken w&h the Aminco Bowman s~~~hotoffuorometer (No. 4-21202)with the phosphorous at&&merit (No. C 27-62140) and-a xe&n lamp (No.
901 Gl; all items manufactured by American Instrument Company, Inc., Silver Spring, Maryland). All studies were made with the slit programme: A 4 mm, B 3 mm, C 3 mm, D 4 mm and E 3 mm. All ;F were recorded with an Aminco X-Y recorder (No. 1620-827, American Instrumeut Company, Reagents All ~~e~~lit~ were purchased in a growth factor analogue kit (Nutritional Biochemicals ~rporation, Cleveland, Ohio). Absolute ethanol (Union Carbule Corporation) purl5ed as pre-
viously described,” was used as the solvent for all phosphorimetrlc measurements. Stock ethanollc solutions (approximate1 lO-*jV) of each corn ound were prepared. Solutions of lower concentrations were prepared 2:y successive dilution. !I than01 was used as the solvent because it is lnexpensive, can be prepared in a highly pure state, and freexes to a clear, rigid glass at 77%. Procedures Ana&tical curves (logarithm of phosph~e~c
signal uersus logarithm of inaction) were obtained to determine whether a compound was analytically useful, and its lit of detection. The limit of detection was de5nedlQ as the concentration which gave a signal-to-noise ratio of tt/s/&. The noise was estimated to be one 5fth of the range of 5uctuation in the background signal, t was the Student t, and n was the mnnber of combined blank and sample measurements made at the limit of detection. With n equal to six and a 99 % con5dence level, the signal-to-noise ratio is approximately 2, The lifetime,r, was measuredby terminating the exciting radiation with a mamnd shutter and plotting the phosphorhnetric signal aemus time wtth the X-Y recorder. The response of the recorder prevented any r hornets shorter than O-5sec. RESULTS AND DISCUSSION Seventeen antlmetabolites having analytically useful phosphorescence are given in Table I. In addition to these 17 compounds, barbituric acid, gamma hexachloro benxene, Dr.,-~fluorophenylalanine, pyrldme-3-&phonic actd, @-2-thienylala&ne, B-2-thlenylseriue, and DL-,8-%hif@alanine phosphoresced, but too weakly to be considered analytically useful. The remaining 12 authnetabolltes didnot phosphoresce [ally1 lyclue, aminoacetic acid @ycine), r+-arglnlne hy~~~o~~, tasparagine, nr.-leucme, n&u&e, ~mnethionim L-canavauine sulphate, %esthlobiotin, ~~y~hol~te~l, sulphoxlde, Dr.-a-methylglutamic acid and sodium salt of pantoyl taurinel. The limits of detection of the antimetabolites listed in Table I compare favourably with those attained by currently accepted calorimetric (0*1-l &ml) and enxymlc (O-0141 @ml) procedures. Previous studies of conjugated rlug system analogues showed that phosphorecence could be enhanced 407
408
short commuIlications
TABLBI.-PHOSPHO RIMmmc cHARAt2lxusn
CSOFSW3RALANTIMBT
ABOLlT@S
Excitation maximum,*t
Emission maximum*t,
Limit of detection,
Lifetime:
Antimetabolite
nm
nm
Pglml
set
3-Acetylpyridine Adenosine 2-Amine-4-methylpyrimidine L-3-3Aminotyrosine dihydrochloride 8-Azaguanine Benzimidazole 2-Chloro-4aminobenzoic acid Desoxypyridoxine hydrochlonde 2,6-Diaminopurine sulphate E%radiol Oxythiamine hydrochloride IX+-Phenyl-lactic acid c+Picolinic acid hydrochloride Pyr~re+&sulphonic acid
277 280 302
:; 438
:::o 0.033
286 282 280
398 442 406
0.81 0.30 0.0059
2.4 ;:;
loas lO?j lol§
312
447
0.069
1.0
10’
290
442
0.076
1.4
lo”
294 292 272 262
424 403 460 383
278
400
0014
5.2
101
272 343 297 312
408 480 411 432
4.8 0.30 0.012 0.0038
1.2
lO?j lP§ 1058 lW§
Quercetin Sulphanilamide 2-Thiouracil
Concentration range of near linearity
CO.5
::;9 ::;
;:;
1.7 2.0 <0*5 5.4
;:;
10‘8 10’1 lO?j
lO?j 1oq IO”8 1oq
* Peaks are uncorrected for instrumental characteristics. t Relative error of f5nm in wavelength of peaks. $ Limiting value of lifetime (0.5 set) is determined by recorder response time. 0 Up r concentration (lO-*M) is still on linear portion of analytical curve. Therefore actual range wt*E”be greater than that reported. by selective substitution on the ring(s) with corresponding improvement of the liits of detection. Therefore, values for limits of detection found in Table I indicate promise and need for further work on analogues of pharmacological interest. Acknowle@exrex~-This work was carried out as part of a study on the hosphorimetric analysis of drugs in blood and urine, supported by the U.S. Public Health Service PGM-11373-05). Department of Chetitry University of Rorida Gainesville, Floriala 32601, U.S.A.
L. B. SANDBRS J. J. Cxroax~~~ J. D. WINEFORDN~R
SummarJr-Phosphorescence excitation and emission wavelength peaks, lifetimes, limits of detection, and concentration ranges of analytical usefulness of 37 antimetabolites in rigid (77’K) ethanolic solution were determined. Seventeen of the metabolites produced analytically useful phosphorescence, whereas the remaining 20 were of limited or no analytical use. ZusammeafaaPrmg-Die We&m&en der Anregungs- und Emissionsmaxima der Phosphoreszenz, Lebensdauem, Nachweisgrenzen und analytisch nutzbare Konzentrationsbereiche von 37 Antimetaboliten in glasartiger (77°K) %thanolischer Ltisung wurden ermittelt. Siebzehn der Metaboliten zeigen analytisch nutzbare Phosphoreszenz, die Ubrigen 20 waren von beschranktem oder keinem analytischen Nutzen.
Short c4mmlmications
409
R&tun&On a d&rmln6 les longueurs d’onde des maximums d’excitation et d’&nission de pho horescen ce, les d&es de vie, les limites de d6tection et les domaines 7 e concentration d’utilite analytique pour 37 antim6tabolites en solution 6thanolique rigide (77°K). Dix sept des m6tabolites produisent une phosphorescence analytiquement utile, tandis que les 20 restant sont d’emploi analytique limit6 ou nul. REFERENCES 1. J. D. Wineforclner, W. J. McCarthy, and P. A. St. John, Phospharimetry as an Analytical Approach in Biochemistry, in Metbo& of Biochemical Analysis, D. Glick, editor, Vol. 15, Interscience. New York, 1967. 2. J. D. Winefordner and H. A. Moye, Anal. Chim. Acta, 1965,32,278. 3. J. D. Winefordner and H. W. Latx, Anal. Cbem., 1963,35,1517. 4. J. D. Winefordner and M. Tin, Anal. Cbim. Acta. 1964,31,239. 5. H. C. Hollitield and J. D. Winefordner, ibid., 1966,36,351. 6. I&m, TaIanta, 1965, 12,860. 7. R. H. Stille and A. Srent-Gyorgyi, Proc. Natl. Acad. Sci. U.S.A., 1957, 43,477. 8. H. A. Move and J. D. Wmefordner. - J. Apr. Food Chem.._ 1965.13,516. . Y
9. Idem, ibid:, 1965, 13,533.
10. J. D. Winefordner, W. J. McCarthy, and P. A. St. John, Anal. Chem., 1967,39,1495.
Tdmta, 1969,Vol. 16,pp. 409to 412. permmonPress. primedin NorthernIdand
Separation of zirconium by thin-layer chromatograplty (Received 20 May 1968. Accepted 29 .Wy 1968) RBWRSD-PHASE thin-layer chromatography has been used for the separation of xirconlum,lJ but normal-phase thin-layer chromatography has scarcely been used. Galiardi and Pokomp separated Zr(IV), Ni(II), Al(m), Ti(IV), Mn(II), Cd(n), V(V), IJO, Fe0 and ZnO by development with acetone-6M hydrochloric acid-dioxan or ethyl methyl ketone on silica 1 layers. Breccia and Spalletti6 separated zirconium-95 and niobium-95 by eluting with methano K” -lOM hydrotbroric acid (25:l) on silica gel layers. Moghissi@separated these radionuclides by eluting with 025M oxalic acid and O-lit4 hydrochloric acid in ethyl methyl ketone, dioxan and water (5:l:l) on Kieselguhr or Kieselgel layers. The present work was, therefore, undertaken to provide more simple and effective TLC systems for the separation involving Zr. SC.Y. Th. U(VI), rare earth and other metal ions. Zirconium remains at the starting point on s&a gel-cellulsoe (5: 1) plates in mesityl oxide-ethanol-nitric acid media, whilst 19 of the other metal ions tested have lamer R, values. extent silver. which tails. This forms the basis of a very selective method for the +ation of zircon&i. ’
EXPERIMENTAL Reagents Test solutions of metal ions, @lM. Appropriate amounts of the nitrates of IJO?), Th, Al, Pb,
Co(B) and Ag were dissolved in 1M nitric acid. Appropriate amounts of the oxides or carbonates of Fe(III), Zn, Cd, Cu(II), Ni, Sc, Y, La, Sm, In, Bi(III), Hg(II), Mg and Ca were converted into the nitrate by treatment with excess of nitric acid, followed by evaporation to dryness. The resulting nitrates were dissolved in 1M nitric acid. Zirconyl chloride was dissolved in dilute nitrrc acid and the solution made ammoniacal. The precipitate of zirconium hydroxide was filtered off and washed free from chloride with demineralized water, and dissolved in nitric acid. The solution was evaporated to dryness and the residue dissolved in 1M nitric acid. Preparation of thin-layer plates. Silica gel (25 g, WAKOGEL B-O, Wako Pure Chemical Industries Co. Ltd., Osaka, Japan) purified according to Seiler’s method,’ and cellulose powder MN-300 (5 g, Machery , Nagel Co., Ltd., Dtlren, Germany) were mixed thoroughly with 87 ml of demineralired water. The slurry was spread 250 p thick on 100 x 100 mm or 200 x 200 mm glass plates. The thin-layer plates were dried first in air for 1 hr and then in an oven at 105-110” for 1 hr and were stored in a desiccator till used.