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ANALYTICAL CHEMISTRY. VOL. 51, NO. 11, SEPTEMBER 1979
benzene dissolved in 1 pL of methanol. Three different integration times were used: 320 320 ms, 320 ms, and 160 ms. The signal-to-noise ratio decreased significantly with shorter integration times and the chromatographic resolution increased as expected. The high noise level is due mainly to the photodiodes. Figures 5a-5c show the partial mass spectra from the total ion current peaks 1-3 in Figure 4a. Peak 1 and 2 represent unknown impurities with base peaks at m / z 281 and 283, respectively, and peak 3 shows the characteristic pattern of hexachlorobenzene. Three different purified solvents of methanol were tested, and all of these showed impurities especially for masses below m / z 250. From our experience with the GC/MS and EOID system, we have found that the solvent used must be carefully purified when substances in the sub-picogram or femtogram range are to be measured. It is also of great importance to use low bleeding columns and to keep a low background in the spectrometer to prevent overloading of the photodiodes. Impurities will limit the full use of the sensitivity in the EOID system when weak peaks from a substance are to be measured. It is also difficult to measure very small amounts of sample using the direct probe inlet since the sample must be dissolved in a solvent before it is introduced into the probe. In LC/MS analysis the difficulties will be increased since a great amount of solvent is introduced into the ion source and ionized. A mass spectrometer with a larger mass range, equipped with an EOID system, including a new type of photodiode arrays, will soon be completed.
+
275
230
275 290
M/Z
V/Z
M/Z
Figure 5. Normalized mass spectrum of the mass range 275-293 of peaks 1-3 in Figure 4a. (a) Peak 1. (b) Peak 2. (c) Peak 3
However, the resolution of the angled CEMA is increased by approximately the same factor. The small mass range covered by the described detector, shows only a part of a mass spectrum and limits the structure information from the cracking pattern of the molecules. However, it can advantageously be used for detection of known compounds. Figure 3a shows the intensity of the total ion current of negative ions in the mass range 275-293 as a result of introducing 100 femtograms (fg) of hexachlorobenzene into the capillary column, kept a t a temperature of 190 "C. The CEMA voltage used was -1.2 kV. An integration time of 320 ms was used to obtain the maximum sensitivity for a single scan of the photodiode in the system used. This is also the approximate maximum integration time that can be used in high resolution capillary column work. Figure 3b shows the three most prominent ions in the detected peak and Figure 3c shows the normalized mass spectrum. Figures 4a-4c show the total ion current in the mass range 275-293 for the introduction of 10 fg of hexachloro-
LITERATURE CITED (1) C. E. Giffin, H. G. Boettger, and D. D. Norris, Int. J . Mass Spectrom. Ion Phys., 15, 437 (1974). (2) . . J. H. Bevnon, D. 0. Jones. and R . G. Cooks. Anal. Chem.. 47. 1734
(1975). (3) D. D. Norris and C. E. Giffin. Proc. SPIEISPSE Techn. Symp. East., 77, 103 (March 1976). (4) H. G. Boettger, G.E. Giffin, D. D. Nonis, W. J. Dreyer, and A. K u p p e m n n , A&. Mass Spectrom. Biochem. Med., 2, 513 (1976). (5) H. H.Tuithof, A. J. H. Boerboom, and H. L. C. Meuzelaar, Int. J . Mass Spectrom. Ion Phys., 17,299 (1975). (6) J. Yinon and H.G. Boettgec, 25th Annual Conference on Mass Spectromeby and Allied Topics, Washington, D.C., May 29-June 3, 1977,paper 711. (7) H. H.Tuithof and A. J. H. Boerboom, Int. J . Mass Spectrom. Ion Phys., 15, 105 (1974).
RECEIVED for review March 28, 1979. Accepted May 8,1979. Work supported by grants from the Swedish Board for Technical Development and from Knut and Alice Wallenbergs Stiftelse.
Quantitative Thin-Layer Chromatography and Mass Spect romet ry of I satinyImethy I Esters of Car boxy Iic Acids G. Gubitz" and W. Wendelin Institut fur Pharmazeutische Chemie der Universitat Graz, A-80 10 Graz, Austria
1-Chlormethylisatin (CMI), a new derivatization reagent for the determination of carboxylic acids by chromatography has been studied. With crown ethers as catalyst, CMI reacts with carboxylic acids under mild reactlon conditions in less than 10 min. The derivatives were separated by TLC and determined in situ by reflectance measurement.
The determination of carboxylic acids by chromatographic methods often requires the use of derivatization reagents. For 0003-2700/79/0351-1690$01.00~0
GC separation, for example, the preparation of the methyl esters (1, 2 ) , benzyl esters ( 3 ) ,pentafluorobenzyl esters (41, and p-bromophenacyl esters ( 5 ) has been reported. Only a few derivatization reagents for enhancement of the sensitivity of detection in TLC or HPLC have been described. Politzer and Griffin (6) used 1-benzyl-3-p-tolyltriazeneto prepare the benzyl esters of carboxylic acids for liquid chromatographic analysis. With 1-p-nitrobenzyl-3-p-tolyltriazene (7), esters with higher UV absorption have been obtained. p-Nitrobenzyl esters have also been prepared using 0 1979 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979 0
CH&
1891
.O
CH2- O-C-R
s
CH; OH
Figure 1.
Scheme of reaction of CMI with carboxylic acids
0-p-nitrobenzyl-N,N'-diisopropylisourea(8) as reagent. Cooper and Anders (9) described the derivatization of carboxylic acids with 2-naphthacyl bromide for HPLC analysis. Durst e t al. (10) prepared phenacyl esters using w,p-dibromoacetophenone with crown ethers as catalysts. Recently, a fluorescent reagent, 4-bromomethyl-7-methoxycoumarinhas also been introduced for TLC (11,12) and HPLC analysis (13) of carboxylic acids. In a previous paper Wendelin and Knotz (14) described a new reagent, chlormethylisatin (CMI) for derivatization of organic acids and reported the melting points and Rrvalues for a number of derivatives. In this paper the application of this reagent for densitometric determination of a series of carboxylic acids is reported. T o reduce the reaction time, crown ethers were used as catalysts. EXPERIMENTAL Apparatus. The measurements on TLC plates were carried out with a Zeiss Chromatogram Spectrophotometer PMQ I1 equipped with a Servogor stripchart recorder and a Spectraphysics Integrator M2. Molar absorptivity measurements were made with a Beckman UV Spectrophotometer Model Acta C 111. The NMR spectra were recorded on a Perkin-Elmer 90-MHz NMR Spectrometer Model R 32: the mass spectra were obtained with an AEI MS-20. Reagents. All solvents and chemicals were of analytical grade. DMF was dried over molecular sieve and distilled. Dibenzo18-crown-6was purchased from Merck. Chlormethylisatin (CMI) was synthetisized as previously described (14,15). The reference esters were prepared as described in (14). Precoated TLC plates Silicagel 60 FZb4,Merck, were washed twice in chloroformmethanol (1:3) before use. Procedure. The sample containing 20 nmol-2 pmol of the carboxylic acid was dissolved in 25 pL of dry DMF in a conical vial of 1-mL volume. Then 20 pL of a freshly prepared solution of CMI in dry DMF containing a 10-fold molar excess were added, followed by a 50-fold molar amount of finely powdered KHC03 and a 5-fold molar amount of dibenzo-18-crown-6in 5 pL DMF. The reaction mixture was then heated at 50 "C for 10 min. After cooling, 100 pL of water were added to convert excess CMI into hydroxymethylisatin. The solution was then extracted with 100 pL of benzene, and an aliquot of 2 pL was transferred to the TLC plate with a microcap. Development was carried out with benzene-ether (70:30) as elution solvent. After evaporation of the solvent, the spots were scanned at 240 nm. Peak areas were calculated by electronic integration. RESULTS AND DISCUSSION Derivatization Studies. The scheme of the reaction is given in Figure 1. Kinetic studies have been carried out to test the optimal reaction conditions. Only 10 min a t a temperature of 50 "C are necessary for the complete reaction of fatty acids like palmitic acid with crown ether as catalyst. Benzoic acid reacts quantitatively in 5 min. Several catalysts like triethylamine K2C03,KHC03, and K2CO3, or KHCOBin combination with crown ethers were studied. Best results could be obtained with KHC03 in combination with crown ethers. Different solvents such as acetone, ethyl acetate, and DMF were tested. DMF has proved to be the most suitable solvent
I , , \-
200
300
400
nm
Figure 2.
Reflectance spectrum of IM-palmitate
Figure 3.
Mass spectrum of IM-propionate
because of its polarity. After completion of the reaction, the reaction mixture is treated with water to convert excess CMI into hydroxymethylisatin. Spectrometric Studies. In the UV spectrum, absorption maxima occur in methanolic solution a t 240 nm and further maxima a t 300 and 420 nm with lower intensity. The molar absorptivity of isatinylmethyl esters (IM-esters) in methanol a t 240 nm is about 14 500. An absorption spectrum of IMpalmitate recorded directly on the TLC plate is shown in Figure 2. The structure of the derivatives is confirmed by IR, NMR, and mass spectrometry. T o examine the usefulness of mass spectrometry of IMesters for identifying carboxylic acids, fragmentation of IM-propionate, -palmitate, and -benzoate was studied. Fibure 3 shows the mass spectrum of IM-propionate. The molecular ions appeared in the three mass spectra with relative intensities of 8-14. Fragment ions with diagnostic value are found in all cases a t M - 176, representing [RCO]+ ions (base peak in the mass spectrum of IM-propionate and -benzoate, respectively). Large peaks a t M - 204, indicating [R]+ ions appear only in the mass spectra of the esters of propionic acid and palmitic acid. Both [RCO]+and [R]+ions are generated by a-cleavage of the esters. No peaks appeared for [IM-OCO]' ions a t mle 222 (alternative products of 0-cleavage with loss of R.). In the mass spectrum of IM-palmitate, McLafferty rearrangement with @-cleavage(which should produce a fragment at mle 294) cannot be observed, but fragments [CnH2n+l]+and [CnH2n-l]+, typical of unbranched long chains, can be seen a t mle 43, 57, 71, 85, and mle 41, 55, 69, 83, respectively. The base peak in the mass spectrum of IM-palmitate a t m / e 160 may be due to methylisatin cations resulting from cleavage of RCOO- and capture of He from the molecular ion. Ions characteristic of all IM-esters at m / e 160, 146, 132, 105, and 77 may result from cleavage or' RCOO-, RCOOCH2, RCOOCH2N., RCOOCH2NCO-, and RCOOCH,NCOCO., respectively, from the molecular ions. These fragments can
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
Table I. Fragmentation of IM-Esters of Carboxylic Acids ( m / e Values and Relative Intensities) M' M' M' ester [MI' [RCO]' [R]' RCOO. t H. RCOO. RCOOCH, propionate 233 (14) 57 (100) 29 (91) 160 (7) 146 (39) palmitate 415 (11) 239 (5.5) 161 (100) 160 (3) 146 (9) benzoate 281 (8) 105 (100) 77 (43) 160 (2) 146 (13)
[PhCO]'
[Phi'
105 (10) 105 (7)
77 (23) 7 1 (4.5)
see RCO'
see R +
Table 11. R f Values of IM-Esters of Carboxylic Acids A. Aliphatic carboxylic acids
Figure 4. Chromatogram of three IM-esters. Elution solvent, benzene-ether (70:30);measure wavelength, 240 nm; scan speed, 50 mm per min. (1) Hydroxymethylisatin, (2) IM-acetate, (3) by-product, (4) IM-butyrate, (5) IM-caprate
formic acid acetic acid propionic acid butyric acid valeric acid caproic acid caprylic acid capric acid lauric acid myristic acid palmitic acid stearic acid
0.29 0.31 0.40 0.43 0.46 0.48 0.52 0.54 0.56 0.57 0.58 0.59
B. Unsaturated acids
acrylic acid oleic acid linoleic acid linolenic acid arachidic acid cinnamic acid
0.38 0.55 0.56 0.56 0.57 0.46
C. Aromatic acids benzoic acid chlorbenzoic acid salicylic acid acetylsalicylic acid
0.48 0.50 0.44 0.33
D. Heterocyclic acids
nicotinic acid biotin
0.04 0.40
Table 111. Yields of the Reaction of Some Carboxylic Acids with CMI (Theoretical amount: 1pg/spot) mean peak area ( x 10) reaction authentic yield, RSD, % acid product sample 7% n=6 J
Figure 5. Chromatogram of four IMesters. For conditions see Figure 4. (1) Hydroxymethylisatin,(2) IM-acetate, (3) by-product,(4) IMvalerate, (5) IM-caprylate, (6) IM-palmitate be attributed to isatinylmethyl, isatinyl, C6H6COC0,benzoyl, and phenyl cations. For relative intensity values see Table I. Chromatographic Studies. The Rf values of some IMesters of organic acids are given in Table 11. The chosen solvent system is suitable for the separation of most of the derivatives. Figures 4 and 5 show chromatograms of mixtures of some organic acids after reaction with CMI. The derivatives of unsaturated fatty acids as oleic, linoleic, and linolenic acid could not be resolved in this system. A good separation of these derivatives, however, can be achieved by HPLC (16). Because CMI shows a strong tailing on the chromatograms, it is necessary to convert it into hydroxymethylisatin after reaction by treatment with water. The spots of all derivatives are well separated from hydroxymethylisatin (Rf0.09). Two by-products with Rf values 0.29 and 0.36 (peaks 2 and 3 in Figure 6), formed in small amounts by decomposition of CMI, are interfering with the quantitative determination of formic and acetic acid only if low amounts of these compounds are to be determined. A good stability of the derivatives was observed. Measurements of the plates after 24 h showed no significant loss in absorption of the spots. Quantitative Results. The time for complete reaction was investigated by comparing peak areas of the reaction products of several acids after reaction times from 2 to 45 min with the peak areas of theoretical amounts of authentic samples on the same TLC plate. Ten minutes were found to be sufficient reaction time. The yields of the reaction of some carboxylic
propionic palmitic oleic benzoic
6768 4522 3845 9082
6792 4612 3980 9091
99.6 98.0 96.6 99.9
1.5 1.7 2.0 1.4
1
I t
4
, 4 Figure 6. Scan of 50 nglspot of IM-palmitate. For conditions see Figure 4. (1) Hydroxymethylisatin, (2, 3) by-products, (4) IM-palmitate
acids with CMI are given in Table 111. The linearity of the calibration curves was excellent over a concentration range of more than one decade. The correlation coefficients were between 0.994 and 0.999. Using the data pair technique (19,i t is possible to obtain a reproducibility corresponding to a relative standard deviation of less than 2% (for amounts of about 1 bg per spot). The detection limits measured at a 3:l signal-to-noise ratio are 20 ng per spot for aromatic carboxylic acids and 50 ng for aliphatic carboxylic acids (Figure 6). These detection limits can be lowered to about a fifth using HPTLC plates. The application of this derivatization method to HPLC is now under investigation. ACKNOWLEDGMENT U. Zeipper is thanked for helpful technical assistance.
ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
LITERATURE CITED
1693
(11) W. Dunaes. Anal. Chem.. 49. 442 (1977). (12) w. &,.A. eyer,. IK Male;, M. Ma&, k. Pietschmann, a. plachetta, R. Sehr, and H. Tuss, Fresenius' 2. Anal. Chem., 288, 361 (1977). (13) W. Dunges and N. Seiler, J . Chromatogr., 145, 483 (1978). (14) W. Wendelin and F. Knotz, Monatsh. Chem., 103, 1632 (1972). (15) F. Knotz, Sci. Pharm., 38, 227 (1970). (16) G. Gubitz, in preparation. (17) H. Bethke, W. Santi, and R. W. Frei, J. Chromtcgr. Sci., 12, 392 (1974).
A. Wnert and K. H. Wssler, Fresenbs' 2.Anal. Chem., 267, 342 (1973). W. Dunges, Chromatographia, 6, 196 (1973)., U. Hintze, H. Roper, and G. Gercken, J . Chromatogr., 67, 481 (1973). H. Ehrsson, Acta Pharm. Suecica, 8, 113 (1971). E. 0.Umeh, J . Chromatogr., 56, 29 (1971). 1. R. Politzer, G. W. Griffin, E. J. Dowly, and J. L. Laseter, Anal. Left:, 6, 539 (1973). Regis Chemical Company, Morton Grove, Ill. D. R. Knapp and Sh. Krueger, Anal. Lett., 8, 603 (1975). M. J. Cooper and M. W. Anders, Anal. Chem., 46, 1849 (1974). H. D. Durst, M. Milano, E. J. Kikta, Jr., S. A. Conneliy, and E. Grushka, Anal. Chem., 47, 1797 (1975).
RECEIVED for review February 14, 1979. Accepted June 4, 1979.
Estimation of High Pressure Liquid Chromatographic Retention Indices John K. Baker Department of Medicinal Chemistry, School of Pharmacy, University of Mississippi, University, Mississippi 38677
time may vary several orders of magnitude because of changes A method for the prediction of the retention times on CIB in the composition of the mobile phase ( 3 ) . It has also been reverse phase columns is developed and applied to a series observed that the substitution of a cyano reverse phase column of propranolol, anthranilic acid, and barbiturate analogues. The for a reverse phase column has little effect on the retention retention properties of the drugs are measured uslng a retention index of most compounds even though the act,ual retention index scale that is based on the relative retention of a series times on the two columns were quite different. Because of of 2-keto alkane standards. The retention index ( I ) of the test these properties, the retention index scale is very useful in compounds is estimated using the equation I = 2007~i- Ire, providing a uniform basis for reporting retention data and for where Ire, is the observed retention index of one reference correlating chemical structure with retention properties. compound that is structurally related to the other compounds The retention index scale was also constructed in such a and 7~ is the sum of the Hansch substituent constants for the manner that most probably it would be linearly related to the test compound. lipophilicity of the compound. Since the lipophilicity of drugs and other compounds can also easily be estimated in a linear manner ( 4 ) , it was anticipated that the two concepts could be combined to form a very simple method for the prediction of the HPLC retention properties of a compound based on an analysis of its chemical structure.
One of the major difficulties in any chromatographic method is the prediction of the retention time or retention volume of a new compound or a derivative of an old one. In the area of gas chromatography, the Kovats retention index scale has become widely used to standardize the reporting of GLC data and as a tool for the correlation of GLC properties and chemical structure ( I , 2). In the area of high pressure liquid chromatographic (HPLC) analysis, qualitative estimations of the retention times of compounds can be readily made for both adsorption and partition columns. However, little progress has been made in the development of methods for the precise prediction of the retention times based simply on the chemical structure of the compound. Recently a retention index scale suitable for use with reverse phase (fi-Bondapak c18 and fi-Bondapak CN) has been reported (3). The retention index scale is based on the relative retention times of a series of 2-keto alkanes. By definition, acetone is given a value of 300 and 2-butanone, 400; etc. A given column-solvent combination is calibrated by chromatographing the 2-keto alkane standards (C3-CZ3) and correlating the logarithm of the observed capacity factors in a linear manner with the defined retention indices. The retention index of a given drug or other test compound is then obtained by a mathematical interpolation between the values of the 2-keto alkane standards. I t has been found that the retention index of a given compound remains nearly the same even though its retention 0003-2700/79/035 1-1693$01.OO/O
EXPERIMENTAL Chromatographic Conditions. A 3.9 mm i.d. X 30 cm CIS reverse phase column (fi-Bondapak CIB,Waters Associates Inc.) with a 10-pm particle size was used for the study. The mobile phase flow rate was 2.0 mL/min and was prepared using 6.6 g K2HP04,8.4 g KH2P04,1.6 L CH30H, and 2.4 L HzO. The pH of the mobile phase was 7.0 before the addition of CH30H. A Waters Associates Inc. M-6000 pump, U6K injector, and Model 440 dual wavelength ultraviolet detector (254 nm and 280 nm) were used. Though the dual wavelength detector was not essential, measurements of the 254 nm/280 nm absorbance ratio greatly facilitated the identification of the drugs in the mixture with the 2-keto alkane standards (5). Materials. The 2-keto alkane standards (C3-C& were obtained from Analabs and the barbiturates from the Theta Corporation. The anthranilic acid derivatives were synthesized (6) by R. F. Borne, Department of Medicinal Chemistry, School of Pharmacy, University of Mississippi. The propranolol analogues were synthesized in these laboratories (7). The methanol used in the mobile phase was freshly distilled while all other chemicals were of reagent grade and were used as obtained. Measurement of Retention Indices. The capacity factor (12') of the drugs and standards were determined from the observed retention time ( t R ) using Equation 1. k'= (tR-tO)/tO
(1)
The retention index of a given 2-keto alkane standard was by
C
1979 American Chemical Society
