- Email: [email protected]
Prostaglandin determination with fluorescent reagents
Prostaglandins Leukotrienes and Medicine14: 25-40,1984
:: )
PROSTAGLANDIN DETERMINATION WITH FLUORESCENT REAGENTS
Reinhold Wintersteiger1) and Heinz Juan*) 'IInstitute of Pharmaceutical Chemistry, 2)Institute for-Experimental and Clinical Pharmacology, Karl-Franzens Universitst Graz, A-8010 Graz, Austria (reprints requests to R.Wintersteiger). ABSTRACT Fluorescent derivatives of PGE2 as a model substance were formed with naphthyl isocyanate, anthracene isocyanate, 4-(6-methylbenzothiazol-2-yl)phenyl isocyanate, benzocoumarin-3-carboxylic acid chloride and 4-bromomethyl-7-methoxycoumarin. The optimum conditions for derivatization with these reagents were investigated and the results were compared. Quantitative determination was performed by means of fluorodensitometric measurement of the derivatives. Using 4-bromomethyl-7-methoxycoumarin as a reagent as little as 100 pg of the PGE2 derivative can be detected. INTRODUCTICN The rapid development in prostaglandin (PG) research during the last years is in part related to the high standard of modern analytical methods. Criteria for such procedures are high sensitivity, selectivity, simple handling and good reproducibility. In particular, high sensitivity is most important for the quantitation of PGs present in very small amounts in biological materials. Therefore, radioimmunoassay (I-31, bioassay (4-6) and gas chromatography-mass-spectrometry (7) are the most commonly employed assay techniques in PG research although each method has its own drawbacks. In addition to its often extremely high sensitivity, radioimmunoassay has the great advantage of a large sample capacity. On the other hand the use of radioactive isotopes and time-consuming separative pretreatment sometimes may be regarded as disadvantages. Using bioassay the sample must be purified extensively before analysis and the results often show'low specifity. Gas chromatography-massspectrometry is a highly specific and sensitive method which :: ) Ded cated to Prof.Dr.E.Schauenstein on the occasion of his 6Sti birthday.
25
can also give structural information. However, its major drawbacks are its low sample capacity and the requirement of expensive equipment. Determination of PGs in the UV-range following conversion to p-nitrophenacyl esters has been described (for review see 8) whereas there are only few references on derivatization with fluorescent reagents (9,101 in spite of the high sensitivity of fluorometric analysis. PGs do not fluoresce so that the formation of fluorophores can be obtained only by reaction with appropriate fluorescent reagents. Using appropriate reagents, a specific functional group of a PG undergoes reaction with agood selectivity. The two reagents known till now are 4-bromomethyl-7-acetoxycoumarin (Br-Mac) (9) and 4-bromomethyl7-methoxycoumarin (Br-Mmc) (IO). Br-Mac also has been used for determination of carboxylic acids (II). It reacts with PGs to form the ester derivatives. Each labeled substance eluted by high performance liquid chromatography (HPLC) is successively hydrolyzed to the fluorescent coumarin derivative to detect this fluorophore quantitatively. Br-Mmc also reacts with the carboxylic groups of PGs to form the corresponding fluorescent esters. These products can be formed in a single step preparation within a few minutes. The products formed from Br-Mmc and PGD2, PGE2, 6-keto-PGF1, and PGF2q can be separated from each other in a single run both by TLC and HPLC (12). However, the minimum detectable amount of PGE2 derivatized and applied to a TLC-plate was only 40 ng. Since hydrolysis is unnecessary with this reagent, optimization of the procedure was performed only with Br-Mmc. The fluorescence quantum yield is rather high so that an improvement of the detection limit appeared to be possible at least by a factor 10. In previous papers (13-16) we reported on the derivatization of compounds containing an isocyanate reagents. alcoholic group with fluorescing The present report describes the possibility of quantitative determination of PGs by means of derivatization with naphthyl isocyanate (NI) , anthracene isocyanate (AI), 4-(6-methylbenzothiazol-2-yl)phenyl isocyanate (Mbp), benzocoumarincarboxylic acid chloride (Bee)and Br-Mmc.Evaluation of the corresponding fluorescent derivatives is carried out fluorodensitometrically. The aim of the present investigation was to determine the optimum reagent under optimum reaction conditions with PGE2 as a model substance. MATERIAL AND METHODS Materials: Naphthyl isocyanate (NI) , triethylendiamine, sodium carbonate and the solvents (benzene, diethylether, ethanol, acetone, methanol, acetonitrile) were of analytical reagent grade from Merck (Darmstadt, GFR) and were used without further purification. Anthracene isocyanate (AI) was synthesized by the reaction of anthracene-2-amine and phosgene following the method of Fieser and Creech (17). 4-(6-methylbenzothiazol-2-yl)phenyl
26
isocyanate (Mbb) was obtained from Sandoz (Basel, Switzerland), Prostaglandin E2, D2 and Faa were a gift from Dr. J. Pike (Upjohn, Kalamazoo, USA) and Dr. B.A. Schblkens (Hoechst, AG, (Br-Mmc) was Frankfurt/M, GFR). 4-bromomethyl-7-methoxycoumarin purchased from Serva (Heidelberg, GFR). 18-crown-6, dibenzo-18crown-6- and diethylamine were from Fluka (Buchs, Switzerland). Xylene was used in UAB IX-quality, dried with sodium. For derivatization attempts conical reaction vials (teflon capped, screw top) from Macherey-Nagel (Diiren,GFR) with a volume of 0.3 ml were used. Benzocoumarincarboxylic acid chloride (Bee) was obtained from the Department of Organic Chemistry, University of Graz. Thin-layer
chromatography:
The chromatographic separations were first performed on TLCplates MN SIL'G with fluorescence indicator, 20 x 20 cm, from Macherey-Nagel, on TLC-plates silica gel G F254 20 x 20 cm, and on RP 18 plates, IO x 10 cm both from Merck. The chromatograms were developed in the following solvent systems: benzenediethylether-ethanol = 50 + 50 + 5 (solvent system I), chloroform-benzene-ethanol-acetic acid = 45 + 15 + 10 + 2 (solvent system 21, methanol-water = 90 + 10 (solvent system 31, chloroform-benzene-ethanol = 35 + 15 + 5 (solvent system 41, chloroform-benzene-ethanol = 45 + 15 + 5 (solvent system 5). Preliminary experiments were done with Macherey-Nagel plates. Since the fluorescence intensity of the derivatives formed was always weaker on these plates only Merck plates were used for experiments described (see table 1). Development was carried out over a distance of 14 cm (for silica gel plates) and 7 cm (for RP plates), respectively. 2 ul were spotted into the plates using 2 ul microcaps. Spots were marked by viewing under a Camag UV lamp. To enhance fluorescence intensity and stability five different dipping solutions were tested: Polyethyleneglycol 4000-ethanol = 1 + 1 (dipping solution I) ; glycerol-ethanol = 1 + 1 (dipping solution II) ; paraffin.liqu.-n.hexane = 4 + 1 (dipping solution III); triethanolamine-3% H202-ethanol = 3 + 3 + 2 (dipping solution IV); triethanolamine-acetone = 1 + 10 (dipping solution V). Instrumentation: The fluorodensitometric analyses were performed on a Perkin Elmer MPF 44 spectrofluorimeter with TLC accessory. The peak areas were integrated with the Perkin Elmer Minigrator M2 after recording the signals with the Perkin Elmer recorder PE 56. The slit settings for quantitative measurement carried out in the energy mode were 10 nm for excitation and 16 nm for emission To some extent the Zeiss Spektrophotodensitometer
27
PMQ II ad-
apted for fluorescence measurement 542 were used. Derivatization
and the Goerz recorder RE
with AI and NI
The solution of PG to be derivatized (1 nmol) was concentrated to dryness under nitrogen in a 0.3 ml conical vial. After adding an equimolar amount of triethylendiamine in 10 ~1 sodium-dried xylene and a 30-fold molar excess of NI in 80 ul xylene, the solutionwas heated in a drying oven for 30 minutes at 950C. Thereafter a two-fold molar excess of diethylamine in 25 ~1 xylene related to the isocyanate concentration was added. The reaction mixture was shaken for about 30 seconds and centrifuged for 5 minutes at 3000 rpm. 2 ul of the supernatent clear solution were spotted on the thin layer plate and development was carried out in the appropriate solvent system in vapor phase saturated chambers. Derivatization
with Mbp
The PG solution was concentrated to dryness in a conical vial as described. 10 ~1 of the equimolar amount of triethylendi; amine in xylene were added. After heating for 2 hours at 50 C, 10 ul xylene containing a 2-fold molar excess of diethylamine related to the isocyanate concentration were pipetted to the reaction mixture. This mixture was shaken and further treated as described. Derivatization
with Bee
After concentrating the PG to dryness, 10 ul xylene containing a 0.3 molar amount of triethylenediamine were pipetted into the 0.3 ml conical vial. A 3-fold molar excess of Bee was added and the reaction mixture was heated in a drying oven for 1 hour at 500C. Further treatment as described. Standard preparations Derivatives of PGE2, PGD2, PGF2o and cetanol were prepared according to the corresponding procedure. PCs and cetanol were dissolved in methanol (1 mg/ml). From the stock solutions aliquots between 0.5 and 500 ng/2 ul were introduced in conical vials for providing a calibration graph. The solvent was removed and derivatization was performed. RESULTS In the case of derivatization with isocyanate, the utilisation of the reagents was tested using cetanol because the urethane of this alcohol was available (13-16). Formation of the urethane of PGE with NI was investigated at reaction temperatures of 20, 50 an3 95OC. Reaction was terminated after 5,10,15,20,
28
30,40 and 60 min. Two ~1 of the reaction mixture were spotted on the TLC plate and developed in the solvent system 1. For reaction kinetics, the 60 min value was started at the time zero, the 40 min value at 20 min (etc.) and the 5 min value last at 55 min so that the reaction was finished at the same times. Thus all the formed derivatives can be spotted at the same time on the TLC plate. Doing so, a possible influence of oxygen or of the silica gel matrix on the fluorescence behaviour is similar for all the derivatives. Optimum reaction rate of NI urethanes is obtained after 25 min at 95oC. To produce a reliably detectable reaction product, the reaction time had to be prolonged to 50 min using 5OoC as a reaction temperature. However fluorescence intensity was considerably lower than at 95OC. Fluorodensitometric measurement was performed at an excitation wavelength of 310 nm and an emission wavelength of 355 nm. Maximum sensitivity was 5 ng. This limit of the detection could not be improved by spotting the derivatives on TLC plate with concentration zone. Correlation of concentration with peak area was given from maximal sensitivity to 300 ng.
RUN (CM)
Figure 1. Chromatogram of the urethane formed from PGE2 with AI. A: blank; B: urethane: solvent system: benzene-diethyletherethanol (50 + 50 + 5). Optimum formation of anthracene urethanes was achieved after 30 min at 5OoC. In addition, a second reaction product was generated showing the identical Rf-value as one of the two peaks of the corresponding blanks (fig. 1). Solvent systems 1 and 2 were well suited for the separation of the anthracene urethane of PGE from by-products. Due to higher fluorescence quantum yield o!zAI compared with NI, a better sensitivity of detection was observed. 700 pg of the AI urethane of PGE2 can be quantitatively detected. Two calibration graphs could be set 29
up: one from 700 pg to 2 ng and the second one from 2 ng to
100 ng. Fluorodensitometric measurements were done at the maxima shown in table 2. Upon scanning the excitation spectrum, several excitation maxima can be observed corresponding to the transition states. For quantitative determination, the maxima at the longer wavelength range (at 393 nm or 425 nm, respectively), were used. Using NI or AI as a reagent, reversed phase (RP) material showed no advantage compared with silica gel material. In contrast, using Mbp as a reagent no separation of the derivatives from the by-products was obtained with silica gel plates. However, development with methanol-H20 (70 + 30) (solvent system 3) on RP18-plates led to good results. Derivatization was performed for 2 hours at 5OoC. Higher temperature was not possible since only acetone was used as a reaction medium. Fig. 2 shows linear correlation between the amount of substance and fluorescence as well as a scan of the derivative. The limit of detection of urethane of Mbp with PGE2 was 750 pg. As in the case of AI the reaction behaviour can be visually observed on the TLC plate by means of an UV-lamp at 366 nm. However, the excitation maximum of 380 nm is on the border of the visual range. Quantitative determination is carried out at the excitation maximum of 340 nm (compare table 3).
Table 1. Chromatographic conditions for separation of derivatives formed with PGE2 (see materials and methods).
Derivatization reagents
Rf-value
NI AI Mbp Bee Br-Mmc
0.28 0.21 0.27 0.50 0.17
solventsystem 1
1 3 4 5
plate material silica silica RP-18 silica silica
gel gel gel gel
For fluorodensitometric determination of the Bee derivatives of PGE2r silica gel material is more suitable than RP-material because of the somewhat higher fluorescence intensity. Best results with regard to reaction time and temperature were obtained at 95oC for 2 hours using solvent system 4. The limit of detection for the Bee urethane of PGE2 was 1 ng. Linear correexcitation- and emission maxima are shown in table 3. For recording the spectra, 500 ng PGE2 were derivatized and measured in comparison with plate background (Fig. 3).
30
20
40
60
80
Linearity of the urethane formed from PGE2 with Mbp ; scan of 750 pg of the derivative. Table 2. Linearity by PGE2
and detection
Derivatization reagents
NI AI MbP Bee Br-Mmc
limits of derivatives
linearity (rig)
5 - 300 0.7 - 2 2- 100 0.75 - 2 2 - 100 2 - 100 1 - 40
formed
maximum sensitivity (ng) 5 0.7 0.75 2 0.1
Kinetics of Br-Mmc were performed at 50°C and 95OC for 2 hours. Optimum conditions for derivatization were found at 50°C for 10 min since in this case formation of by-products is very low. Further investigations with regard to different catalysts, molarity of the excess of reagent or of reaction medium did not result in improvements. Because of tailing peaks on RP plates silica gel material was'preferred.As low as 100 pg of the PGE2derivative could be determined quantitatively. In a series of experiments,
a mixture of PGE2, PGD2 and PGF2,
31
h ex
PLATE GROUN
r
_
WI
Figure 3. Excitation of PGE2.
and emission spectrum of the Bee-derivative
was derivatized with Br-Mmc and well separated by TLC (Fig. 4). Quantitative analysis was performed fluorodensitometrically after separation with solvent system 5.
32
I:,
(:I\
0
0
c!
:;
:.;
6 *-
c-’
?? ?? .-’
42
0
0
0
0
0
0
.
.
.
.
.
1
2
3
4
5
Figure 4. TLC of the Br-Mmc derivative of PGE PGD2 and PGF2,. Solvent system: chloroform-benzene-ethanol (4V + 15 + 5). 1: Blank, 2: PGF2,, 3: PGE2, 4: PGD2, 5: mixture. DISCUSSION PGs show three functional groups appropriate for derivatization The carboxylic acid group, the keto group on the cyclopentane ring (except PGF2c 1 and the alcoholic hydroxylic group on the ring and in the side chain. Reaction of alcohols with fluorescent isocyanates to urethanes had beenperformed successfully (13-16). Therefore, first these reagents were tested for derivatization of PGs. The scheme of the reaction and the possible reaction products to be generated are shownin fig. 5. The isocyanates can attack at the hydroxylic group of the side chain or of the ring or at both groups. A reaction of the carboxylic group can be excludedunder the present reactionccmditions with high probability.
33
R-3
R,:
@
R3:
a
R3:
j&j-0-
Figure 5. Reaction scheme of PGE2 with fluorescent isocyanates. NI, AI and Mbp were used as isocyanates. They possess different reactivity and fluorescence quantum yield. Furthermore, difference in polarity leads to different migration behaviour of the derivatives on the plate. To increase intensity and stability of fluorescence of the urethane generated, the developed TLCs were immersed into the dipping solutions listed in the method section. The results were compared with those obtained usins not-immersed plates. In all cases best results were obtained using dipping-solution I which increased fluorescence by about 100%. Fluorescence did not decrease over a period of 10 hours.
34
Table 3. Excitation- and emission maxima of derivatives formed with PGE2 (for explanation see results)
Derivatization reagents
NI AI *P Bee Br-Mmc
Excitation maximum (nm) 310 355 377 393 340 384 338
Emission maximum (nm) 355 425 380
430 390
Because of heat-instability of the PGs, kinetics were carried out at different temperatures. However, it was unclear up to which temperature a decomposition of the PG would occur before derivatization. At room temperature NI does not react to the corresponding urethane within 3 hours whereas AI leads to derivatization. This is surprising since the reactivity of the isocyanates decreases with increasing number of aromates as is well known. Interestingly, the rate of derivatization is far better at 50°C than at a higher temperature. Fig. 6 shows reaction kinetics at room temperature, 50°C and 130°C. The time course of the curve appears to indicate that 2 different products were generated at higher temperature. Even at 10 min there was optimum derivatization (50°C, 13OOC). Thereafter, fluorescence intensity declines quickly to a minimum and again increases up to a maximum at a reaction time of 1 to 2 hours. An exact structuraldefinition of the products generated can be only performed spectroscopically following synthesis, isolation and purification in the mg range. As mentioned in an earlier paper (151, Mbp does not show the expected reactivity although the reactive phenylisocyanate component is present in the molecule. This fact is documented also by acontinuous increase in the rate of derivatization up to the highest time of 3 hours. However, the slope of the curve is rather small (Fig. 7) so that 75% of the maximum fluorescence intensity can be observed after 2 hours. Bee is an absolutely new fluorescence derivatization reagent which is characterized by a high fluorescence quantum yield. For derivatization of a PG with Bee, the carboxylic group is available in addition to the hydroxylic groups. Due to the higher affinity of the hydroxylic group to the acid chloride, first the generation of a fluorescent ester has to be assumed. Thereafter, according to reaction conditions, a derivatization
35
Figure 6. Kinetics of the derivative of AI with PGE2 at room temperature (W 1, 500C (0) and 130°C (A).
20
60
140
700
180
Figure 7. Kinetics of the derivative of Mbp with PGE2 at 50°C. at the carboxylic group can occur. The different derivatizatic products which can be generated are shown in figure 8.
36
Figure 8. Reaction scheme of PGE3 with Bee. As a further reagent for fluorescence Br-Mmc was used. Br-Mmc reacts in the the carboxylic group of the PG thereby to the whole molecule. The reaction is catalyzed by 18-crown-6 The scheme of reaction with PGE3 is presented in figure 9. Due to the high light sensitivity of Br-Mmc it is important to per form all operations without light. Generation of by-products can be further diminished using redistilled p.a. acetone.
37
I
18-Crown-6 K&Q
Figure 9. Reaction scheme of PGE2 with B?Mmc. Summarizing the results it can be concluded that each of the reagents used gave highly fluorescent derivatives with PGE2 as a model substance. Reaction conditions with regard to timedependence, temperature, molarity of reagents or catalyst and chromatographical separation were optimized. Highest sensitivity was obtained with Br-Mmc at a low reaction temperature (50°C) and a reaction time of only 10 minutes. In this case 100 pg can be quantitatively determined. That means an increase in sensitivity by a factor of 200 was achieved compared with that obtained by Turk et al. (12). Derivatization occurs only with the carboxylic group of the PG. Detection of pg-amounts at an acceptable derivatization temperature (50°C) could also be accomplished with AI as a reagent. Derivatization with isocyanates and Bee occurs at the alcoholic hydroxylic groups. The possibility of selecting various reagents allows a derivatization of appropriate functional group on a well informed basis. For derivatization of the alcoholic group 4 different reagents are available which enable the use of silica gel as well as RP material. The high sensitivity appears to present the described procedure as a viable alternative and significant improvement of methods thus far described. This procedure shall be transfered to other PGs in combination with HPLC.
38
REFERENCES 1. Samuelsson B, Granstrom E, Green K, Hamberg M, Hammarstrijm S. Prostaglandins. Annual Reviews of Biochemistry 44: 669, 1975. 2. Samuelsson B, Goldyne M, Granstrijm E, Hamberg M, HammarStrom S, Malmsten C. Prostaglandins and thromboxanes. Annual Reviews of Biochemistry 47: 995, 1978. 3. Granstrijm E. Radioimmunoassay glandins 15: 3, 1978.
of Prostaglandines.
4. Gaddun JH. The technique of superfusion. 8: 321, 1953.
Prosta-
Brit J Pharmacol
5. Vane JR. !l!herelease and fate of vasoactive hormones in the circulation. Brit J Pharmacol 35: 209, 1969. 6. Moncada S, Ferreira SH, Vane JR. Bioassay of prostaglandins and biologically active substances derived from arachidonic acid. Advances in Prostaglandin and Thromboxanes Res 5: 211, 1978. 7. Green K, Hamberg M, Samuelsson B, Smigel M, Frijhlich JC. Measurement of prostaglandins, thromboxanes, prostacyclin, and their metabolites by gas liquid chromatography-massspectrometry. Advances in Prostaglandin and Thromboxane Res 5: 39, 1978. Pryde A, Gilbert MT. Prostaglandins. In: Applications of high performance liquid chromatography, Halsted Press, New York, 1979. Tsuchiya H, Hayashi T, Naruse H, Takagi N. Sensitive high performance liquid chromatographic method for prostaglandins using a fluorescence reagent, 4-bromomethyl-7acetoxycoumarin. J Chromatogr 231: 247, 1982. 10. Diinges W. 4-Bromomethyl-7-methoxycoumarin as a new fluorescence label for fatty acids. Anal Chem 49: 442, 1977. 11. Tsuchiya H, Hayashi T, Naruse H, Takagi N. High performance liquid chromatography of carboxylic acids using 4-bromomethyl-7-acetoxycoumarin as a fluorescence reagent. J Chromatogr 234: 121, 1982. 12. Turk J, Weiss StJ, Davis JE, Needleman P. Fluorescent derivatives of prostaglandins and thromboxanes for liquid chromatography. Prostaglandins 16: 291, 1978.
39
13.
Wintersteiger R, Wenninger-Weinzierl G. Neues empfindliches Verfahren zur diinnschichtchromatographischen Analyse von Substanzen mit alkoholischer Hydroxylgruppe durch Reaktion mit Naphthylisocyanat. Fresenius Z Anal Chem 309:201, 1981.
14.
Wintersteiger R, Wenninger-Weinzierl G, Pacha W. Highperformance liquid chromatography of naphthylurethanes by 'means of fluorescence detection. J Chromatogr 237: 399, 1982.
15. Wintersteiger R, Gamse E, Pacha W. Quantifizierung von alkoholischen Verbindungen und Aminen mit 4-(6-Methylbenzothiazol-2-yl)phenylisocyanat (Mbp). Fresenius Z Anal Chemie 312: 455, 1982. 16. Wintersteiger R. Anthracene isocyanateas anew fluorescence label for wundswith an alcoholic group. J Liqu Chromatogr 5: 897, 1982. 17. Fieser LF, Creech HJ. The conjugation of amino acids with isocyanates of the anthracene and 1,2_benzanthracene series. J Am Chem Sot 61: 3502, 1939.
40
:: )
PROSTAGLANDIN DETERMINATION WITH FLUORESCENT REAGENTS
Reinhold Wintersteiger1) and Heinz Juan*) 'IInstitute of Pharmaceutical Chemistry, 2)Institute for-Experimental and Clinical Pharmacology, Karl-Franzens Universitst Graz, A-8010 Graz, Austria (reprints requests to R.Wintersteiger). ABSTRACT Fluorescent derivatives of PGE2 as a model substance were formed with naphthyl isocyanate, anthracene isocyanate, 4-(6-methylbenzothiazol-2-yl)phenyl isocyanate, benzocoumarin-3-carboxylic acid chloride and 4-bromomethyl-7-methoxycoumarin. The optimum conditions for derivatization with these reagents were investigated and the results were compared. Quantitative determination was performed by means of fluorodensitometric measurement of the derivatives. Using 4-bromomethyl-7-methoxycoumarin as a reagent as little as 100 pg of the PGE2 derivative can be detected. INTRODUCTICN The rapid development in prostaglandin (PG) research during the last years is in part related to the high standard of modern analytical methods. Criteria for such procedures are high sensitivity, selectivity, simple handling and good reproducibility. In particular, high sensitivity is most important for the quantitation of PGs present in very small amounts in biological materials. Therefore, radioimmunoassay (I-31, bioassay (4-6) and gas chromatography-mass-spectrometry (7) are the most commonly employed assay techniques in PG research although each method has its own drawbacks. In addition to its often extremely high sensitivity, radioimmunoassay has the great advantage of a large sample capacity. On the other hand the use of radioactive isotopes and time-consuming separative pretreatment sometimes may be regarded as disadvantages. Using bioassay the sample must be purified extensively before analysis and the results often show'low specifity. Gas chromatography-massspectrometry is a highly specific and sensitive method which :: ) Ded cated to Prof.Dr.E.Schauenstein on the occasion of his 6Sti birthday.
25
can also give structural information. However, its major drawbacks are its low sample capacity and the requirement of expensive equipment. Determination of PGs in the UV-range following conversion to p-nitrophenacyl esters has been described (for review see 8) whereas there are only few references on derivatization with fluorescent reagents (9,101 in spite of the high sensitivity of fluorometric analysis. PGs do not fluoresce so that the formation of fluorophores can be obtained only by reaction with appropriate fluorescent reagents. Using appropriate reagents, a specific functional group of a PG undergoes reaction with agood selectivity. The two reagents known till now are 4-bromomethyl-7-acetoxycoumarin (Br-Mac) (9) and 4-bromomethyl7-methoxycoumarin (Br-Mmc) (IO). Br-Mac also has been used for determination of carboxylic acids (II). It reacts with PGs to form the ester derivatives. Each labeled substance eluted by high performance liquid chromatography (HPLC) is successively hydrolyzed to the fluorescent coumarin derivative to detect this fluorophore quantitatively. Br-Mmc also reacts with the carboxylic groups of PGs to form the corresponding fluorescent esters. These products can be formed in a single step preparation within a few minutes. The products formed from Br-Mmc and PGD2, PGE2, 6-keto-PGF1, and PGF2q can be separated from each other in a single run both by TLC and HPLC (12). However, the minimum detectable amount of PGE2 derivatized and applied to a TLC-plate was only 40 ng. Since hydrolysis is unnecessary with this reagent, optimization of the procedure was performed only with Br-Mmc. The fluorescence quantum yield is rather high so that an improvement of the detection limit appeared to be possible at least by a factor 10. In previous papers (13-16) we reported on the derivatization of compounds containing an isocyanate reagents. alcoholic group with fluorescing The present report describes the possibility of quantitative determination of PGs by means of derivatization with naphthyl isocyanate (NI) , anthracene isocyanate (AI), 4-(6-methylbenzothiazol-2-yl)phenyl isocyanate (Mbp), benzocoumarincarboxylic acid chloride (Bee)and Br-Mmc.Evaluation of the corresponding fluorescent derivatives is carried out fluorodensitometrically. The aim of the present investigation was to determine the optimum reagent under optimum reaction conditions with PGE2 as a model substance. MATERIAL AND METHODS Materials: Naphthyl isocyanate (NI) , triethylendiamine, sodium carbonate and the solvents (benzene, diethylether, ethanol, acetone, methanol, acetonitrile) were of analytical reagent grade from Merck (Darmstadt, GFR) and were used without further purification. Anthracene isocyanate (AI) was synthesized by the reaction of anthracene-2-amine and phosgene following the method of Fieser and Creech (17). 4-(6-methylbenzothiazol-2-yl)phenyl
26
isocyanate (Mbb) was obtained from Sandoz (Basel, Switzerland), Prostaglandin E2, D2 and Faa were a gift from Dr. J. Pike (Upjohn, Kalamazoo, USA) and Dr. B.A. Schblkens (Hoechst, AG, (Br-Mmc) was Frankfurt/M, GFR). 4-bromomethyl-7-methoxycoumarin purchased from Serva (Heidelberg, GFR). 18-crown-6, dibenzo-18crown-6- and diethylamine were from Fluka (Buchs, Switzerland). Xylene was used in UAB IX-quality, dried with sodium. For derivatization attempts conical reaction vials (teflon capped, screw top) from Macherey-Nagel (Diiren,GFR) with a volume of 0.3 ml were used. Benzocoumarincarboxylic acid chloride (Bee) was obtained from the Department of Organic Chemistry, University of Graz. Thin-layer
chromatography:
The chromatographic separations were first performed on TLCplates MN SIL'G with fluorescence indicator, 20 x 20 cm, from Macherey-Nagel, on TLC-plates silica gel G F254 20 x 20 cm, and on RP 18 plates, IO x 10 cm both from Merck. The chromatograms were developed in the following solvent systems: benzenediethylether-ethanol = 50 + 50 + 5 (solvent system I), chloroform-benzene-ethanol-acetic acid = 45 + 15 + 10 + 2 (solvent system 21, methanol-water = 90 + 10 (solvent system 31, chloroform-benzene-ethanol = 35 + 15 + 5 (solvent system 41, chloroform-benzene-ethanol = 45 + 15 + 5 (solvent system 5). Preliminary experiments were done with Macherey-Nagel plates. Since the fluorescence intensity of the derivatives formed was always weaker on these plates only Merck plates were used for experiments described (see table 1). Development was carried out over a distance of 14 cm (for silica gel plates) and 7 cm (for RP plates), respectively. 2 ul were spotted into the plates using 2 ul microcaps. Spots were marked by viewing under a Camag UV lamp. To enhance fluorescence intensity and stability five different dipping solutions were tested: Polyethyleneglycol 4000-ethanol = 1 + 1 (dipping solution I) ; glycerol-ethanol = 1 + 1 (dipping solution II) ; paraffin.liqu.-n.hexane = 4 + 1 (dipping solution III); triethanolamine-3% H202-ethanol = 3 + 3 + 2 (dipping solution IV); triethanolamine-acetone = 1 + 10 (dipping solution V). Instrumentation: The fluorodensitometric analyses were performed on a Perkin Elmer MPF 44 spectrofluorimeter with TLC accessory. The peak areas were integrated with the Perkin Elmer Minigrator M2 after recording the signals with the Perkin Elmer recorder PE 56. The slit settings for quantitative measurement carried out in the energy mode were 10 nm for excitation and 16 nm for emission To some extent the Zeiss Spektrophotodensitometer
27
PMQ II ad-
apted for fluorescence measurement 542 were used. Derivatization
and the Goerz recorder RE
with AI and NI
The solution of PG to be derivatized (1 nmol) was concentrated to dryness under nitrogen in a 0.3 ml conical vial. After adding an equimolar amount of triethylendiamine in 10 ~1 sodium-dried xylene and a 30-fold molar excess of NI in 80 ul xylene, the solutionwas heated in a drying oven for 30 minutes at 950C. Thereafter a two-fold molar excess of diethylamine in 25 ~1 xylene related to the isocyanate concentration was added. The reaction mixture was shaken for about 30 seconds and centrifuged for 5 minutes at 3000 rpm. 2 ul of the supernatent clear solution were spotted on the thin layer plate and development was carried out in the appropriate solvent system in vapor phase saturated chambers. Derivatization
with Mbp
The PG solution was concentrated to dryness in a conical vial as described. 10 ~1 of the equimolar amount of triethylendi; amine in xylene were added. After heating for 2 hours at 50 C, 10 ul xylene containing a 2-fold molar excess of diethylamine related to the isocyanate concentration were pipetted to the reaction mixture. This mixture was shaken and further treated as described. Derivatization
with Bee
After concentrating the PG to dryness, 10 ul xylene containing a 0.3 molar amount of triethylenediamine were pipetted into the 0.3 ml conical vial. A 3-fold molar excess of Bee was added and the reaction mixture was heated in a drying oven for 1 hour at 500C. Further treatment as described. Standard preparations Derivatives of PGE2, PGD2, PGF2o and cetanol were prepared according to the corresponding procedure. PCs and cetanol were dissolved in methanol (1 mg/ml). From the stock solutions aliquots between 0.5 and 500 ng/2 ul were introduced in conical vials for providing a calibration graph. The solvent was removed and derivatization was performed. RESULTS In the case of derivatization with isocyanate, the utilisation of the reagents was tested using cetanol because the urethane of this alcohol was available (13-16). Formation of the urethane of PGE with NI was investigated at reaction temperatures of 20, 50 an3 95OC. Reaction was terminated after 5,10,15,20,
28
30,40 and 60 min. Two ~1 of the reaction mixture were spotted on the TLC plate and developed in the solvent system 1. For reaction kinetics, the 60 min value was started at the time zero, the 40 min value at 20 min (etc.) and the 5 min value last at 55 min so that the reaction was finished at the same times. Thus all the formed derivatives can be spotted at the same time on the TLC plate. Doing so, a possible influence of oxygen or of the silica gel matrix on the fluorescence behaviour is similar for all the derivatives. Optimum reaction rate of NI urethanes is obtained after 25 min at 95oC. To produce a reliably detectable reaction product, the reaction time had to be prolonged to 50 min using 5OoC as a reaction temperature. However fluorescence intensity was considerably lower than at 95OC. Fluorodensitometric measurement was performed at an excitation wavelength of 310 nm and an emission wavelength of 355 nm. Maximum sensitivity was 5 ng. This limit of the detection could not be improved by spotting the derivatives on TLC plate with concentration zone. Correlation of concentration with peak area was given from maximal sensitivity to 300 ng.
RUN (CM)
Figure 1. Chromatogram of the urethane formed from PGE2 with AI. A: blank; B: urethane: solvent system: benzene-diethyletherethanol (50 + 50 + 5). Optimum formation of anthracene urethanes was achieved after 30 min at 5OoC. In addition, a second reaction product was generated showing the identical Rf-value as one of the two peaks of the corresponding blanks (fig. 1). Solvent systems 1 and 2 were well suited for the separation of the anthracene urethane of PGE from by-products. Due to higher fluorescence quantum yield o!zAI compared with NI, a better sensitivity of detection was observed. 700 pg of the AI urethane of PGE2 can be quantitatively detected. Two calibration graphs could be set 29
up: one from 700 pg to 2 ng and the second one from 2 ng to
100 ng. Fluorodensitometric measurements were done at the maxima shown in table 2. Upon scanning the excitation spectrum, several excitation maxima can be observed corresponding to the transition states. For quantitative determination, the maxima at the longer wavelength range (at 393 nm or 425 nm, respectively), were used. Using NI or AI as a reagent, reversed phase (RP) material showed no advantage compared with silica gel material. In contrast, using Mbp as a reagent no separation of the derivatives from the by-products was obtained with silica gel plates. However, development with methanol-H20 (70 + 30) (solvent system 3) on RP18-plates led to good results. Derivatization was performed for 2 hours at 5OoC. Higher temperature was not possible since only acetone was used as a reaction medium. Fig. 2 shows linear correlation between the amount of substance and fluorescence as well as a scan of the derivative. The limit of detection of urethane of Mbp with PGE2 was 750 pg. As in the case of AI the reaction behaviour can be visually observed on the TLC plate by means of an UV-lamp at 366 nm. However, the excitation maximum of 380 nm is on the border of the visual range. Quantitative determination is carried out at the excitation maximum of 340 nm (compare table 3).
Table 1. Chromatographic conditions for separation of derivatives formed with PGE2 (see materials and methods).
Derivatization reagents
Rf-value
NI AI Mbp Bee Br-Mmc
0.28 0.21 0.27 0.50 0.17
solventsystem 1
1 3 4 5
plate material silica silica RP-18 silica silica
gel gel gel gel
For fluorodensitometric determination of the Bee derivatives of PGE2r silica gel material is more suitable than RP-material because of the somewhat higher fluorescence intensity. Best results with regard to reaction time and temperature were obtained at 95oC for 2 hours using solvent system 4. The limit of detection for the Bee urethane of PGE2 was 1 ng. Linear correexcitation- and emission maxima are shown in table 3. For recording the spectra, 500 ng PGE2 were derivatized and measured in comparison with plate background (Fig. 3).
30
20
40
60
80
Linearity of the urethane formed from PGE2 with Mbp ; scan of 750 pg of the derivative. Table 2. Linearity by PGE2
and detection
Derivatization reagents
NI AI MbP Bee Br-Mmc
limits of derivatives
linearity (rig)
5 - 300 0.7 - 2 2- 100 0.75 - 2 2 - 100 2 - 100 1 - 40
formed
maximum sensitivity (ng) 5 0.7 0.75 2 0.1
Kinetics of Br-Mmc were performed at 50°C and 95OC for 2 hours. Optimum conditions for derivatization were found at 50°C for 10 min since in this case formation of by-products is very low. Further investigations with regard to different catalysts, molarity of the excess of reagent or of reaction medium did not result in improvements. Because of tailing peaks on RP plates silica gel material was'preferred.As low as 100 pg of the PGE2derivative could be determined quantitatively. In a series of experiments,
a mixture of PGE2, PGD2 and PGF2,
31
h ex
PLATE GROUN
r
_
WI
Figure 3. Excitation of PGE2.
and emission spectrum of the Bee-derivative
was derivatized with Br-Mmc and well separated by TLC (Fig. 4). Quantitative analysis was performed fluorodensitometrically after separation with solvent system 5.
32
I:,
(:I\
0
0
c!
:;
:.;
6 *-
c-’
?? ?? .-’
42
0
0
0
0
0
0
.
.
.
.
.
1
2
3
4
5
Figure 4. TLC of the Br-Mmc derivative of PGE PGD2 and PGF2,. Solvent system: chloroform-benzene-ethanol (4V + 15 + 5). 1: Blank, 2: PGF2,, 3: PGE2, 4: PGD2, 5: mixture. DISCUSSION PGs show three functional groups appropriate for derivatization The carboxylic acid group, the keto group on the cyclopentane ring (except PGF2c 1 and the alcoholic hydroxylic group on the ring and in the side chain. Reaction of alcohols with fluorescent isocyanates to urethanes had beenperformed successfully (13-16). Therefore, first these reagents were tested for derivatization of PGs. The scheme of the reaction and the possible reaction products to be generated are shownin fig. 5. The isocyanates can attack at the hydroxylic group of the side chain or of the ring or at both groups. A reaction of the carboxylic group can be excludedunder the present reactionccmditions with high probability.
33
R-3
R,:
@
R3:
a
R3:
j&j-0-
Figure 5. Reaction scheme of PGE2 with fluorescent isocyanates. NI, AI and Mbp were used as isocyanates. They possess different reactivity and fluorescence quantum yield. Furthermore, difference in polarity leads to different migration behaviour of the derivatives on the plate. To increase intensity and stability of fluorescence of the urethane generated, the developed TLCs were immersed into the dipping solutions listed in the method section. The results were compared with those obtained usins not-immersed plates. In all cases best results were obtained using dipping-solution I which increased fluorescence by about 100%. Fluorescence did not decrease over a period of 10 hours.
34
Table 3. Excitation- and emission maxima of derivatives formed with PGE2 (for explanation see results)
Derivatization reagents
NI AI *P Bee Br-Mmc
Excitation maximum (nm) 310 355 377 393 340 384 338
Emission maximum (nm) 355 425 380
430 390
Because of heat-instability of the PGs, kinetics were carried out at different temperatures. However, it was unclear up to which temperature a decomposition of the PG would occur before derivatization. At room temperature NI does not react to the corresponding urethane within 3 hours whereas AI leads to derivatization. This is surprising since the reactivity of the isocyanates decreases with increasing number of aromates as is well known. Interestingly, the rate of derivatization is far better at 50°C than at a higher temperature. Fig. 6 shows reaction kinetics at room temperature, 50°C and 130°C. The time course of the curve appears to indicate that 2 different products were generated at higher temperature. Even at 10 min there was optimum derivatization (50°C, 13OOC). Thereafter, fluorescence intensity declines quickly to a minimum and again increases up to a maximum at a reaction time of 1 to 2 hours. An exact structuraldefinition of the products generated can be only performed spectroscopically following synthesis, isolation and purification in the mg range. As mentioned in an earlier paper (151, Mbp does not show the expected reactivity although the reactive phenylisocyanate component is present in the molecule. This fact is documented also by acontinuous increase in the rate of derivatization up to the highest time of 3 hours. However, the slope of the curve is rather small (Fig. 7) so that 75% of the maximum fluorescence intensity can be observed after 2 hours. Bee is an absolutely new fluorescence derivatization reagent which is characterized by a high fluorescence quantum yield. For derivatization of a PG with Bee, the carboxylic group is available in addition to the hydroxylic groups. Due to the higher affinity of the hydroxylic group to the acid chloride, first the generation of a fluorescent ester has to be assumed. Thereafter, according to reaction conditions, a derivatization
35
Figure 6. Kinetics of the derivative of AI with PGE2 at room temperature (W 1, 500C (0) and 130°C (A).
20
60
140
700
180
Figure 7. Kinetics of the derivative of Mbp with PGE2 at 50°C. at the carboxylic group can occur. The different derivatizatic products which can be generated are shown in figure 8.
36
Figure 8. Reaction scheme of PGE3 with Bee. As a further reagent for fluorescence Br-Mmc was used. Br-Mmc reacts in the the carboxylic group of the PG thereby to the whole molecule. The reaction is catalyzed by 18-crown-6 The scheme of reaction with PGE3 is presented in figure 9. Due to the high light sensitivity of Br-Mmc it is important to per form all operations without light. Generation of by-products can be further diminished using redistilled p.a. acetone.
37
I
18-Crown-6 K&Q
Figure 9. Reaction scheme of PGE2 with B?Mmc. Summarizing the results it can be concluded that each of the reagents used gave highly fluorescent derivatives with PGE2 as a model substance. Reaction conditions with regard to timedependence, temperature, molarity of reagents or catalyst and chromatographical separation were optimized. Highest sensitivity was obtained with Br-Mmc at a low reaction temperature (50°C) and a reaction time of only 10 minutes. In this case 100 pg can be quantitatively determined. That means an increase in sensitivity by a factor of 200 was achieved compared with that obtained by Turk et al. (12). Derivatization occurs only with the carboxylic group of the PG. Detection of pg-amounts at an acceptable derivatization temperature (50°C) could also be accomplished with AI as a reagent. Derivatization with isocyanates and Bee occurs at the alcoholic hydroxylic groups. The possibility of selecting various reagents allows a derivatization of appropriate functional group on a well informed basis. For derivatization of the alcoholic group 4 different reagents are available which enable the use of silica gel as well as RP material. The high sensitivity appears to present the described procedure as a viable alternative and significant improvement of methods thus far described. This procedure shall be transfered to other PGs in combination with HPLC.
38
REFERENCES 1. Samuelsson B, Granstrom E, Green K, Hamberg M, Hammarstrijm S. Prostaglandins. Annual Reviews of Biochemistry 44: 669, 1975. 2. Samuelsson B, Goldyne M, Granstrijm E, Hamberg M, HammarStrom S, Malmsten C. Prostaglandins and thromboxanes. Annual Reviews of Biochemistry 47: 995, 1978. 3. Granstrijm E. Radioimmunoassay glandins 15: 3, 1978.
of Prostaglandines.
4. Gaddun JH. The technique of superfusion. 8: 321, 1953.
Prosta-
Brit J Pharmacol
5. Vane JR. !l!herelease and fate of vasoactive hormones in the circulation. Brit J Pharmacol 35: 209, 1969. 6. Moncada S, Ferreira SH, Vane JR. Bioassay of prostaglandins and biologically active substances derived from arachidonic acid. Advances in Prostaglandin and Thromboxanes Res 5: 211, 1978. 7. Green K, Hamberg M, Samuelsson B, Smigel M, Frijhlich JC. Measurement of prostaglandins, thromboxanes, prostacyclin, and their metabolites by gas liquid chromatography-massspectrometry. Advances in Prostaglandin and Thromboxane Res 5: 39, 1978. Pryde A, Gilbert MT. Prostaglandins. In: Applications of high performance liquid chromatography, Halsted Press, New York, 1979. Tsuchiya H, Hayashi T, Naruse H, Takagi N. Sensitive high performance liquid chromatographic method for prostaglandins using a fluorescence reagent, 4-bromomethyl-7acetoxycoumarin. J Chromatogr 231: 247, 1982. 10. Diinges W. 4-Bromomethyl-7-methoxycoumarin as a new fluorescence label for fatty acids. Anal Chem 49: 442, 1977. 11. Tsuchiya H, Hayashi T, Naruse H, Takagi N. High performance liquid chromatography of carboxylic acids using 4-bromomethyl-7-acetoxycoumarin as a fluorescence reagent. J Chromatogr 234: 121, 1982. 12. Turk J, Weiss StJ, Davis JE, Needleman P. Fluorescent derivatives of prostaglandins and thromboxanes for liquid chromatography. Prostaglandins 16: 291, 1978.
39
13.
Wintersteiger R, Wenninger-Weinzierl G. Neues empfindliches Verfahren zur diinnschichtchromatographischen Analyse von Substanzen mit alkoholischer Hydroxylgruppe durch Reaktion mit Naphthylisocyanat. Fresenius Z Anal Chem 309:201, 1981.
14.
Wintersteiger R, Wenninger-Weinzierl G, Pacha W. Highperformance liquid chromatography of naphthylurethanes by 'means of fluorescence detection. J Chromatogr 237: 399, 1982.
15. Wintersteiger R, Gamse E, Pacha W. Quantifizierung von alkoholischen Verbindungen und Aminen mit 4-(6-Methylbenzothiazol-2-yl)phenylisocyanat (Mbp). Fresenius Z Anal Chemie 312: 455, 1982. 16. Wintersteiger R. Anthracene isocyanateas anew fluorescence label for wundswith an alcoholic group. J Liqu Chromatogr 5: 897, 1982. 17. Fieser LF, Creech HJ. The conjugation of amino acids with isocyanates of the anthracene and 1,2_benzanthracene series. J Am Chem Sot 61: 3502, 1939.
40