ACKNOWLEDGMENT
L. Magos and T. W. Ciarkson, J. Assoc. Off. Anal. Chem., 55, 966-971
Appreciation is expressed to T. W. Clarkson and M. R. Greenwood of the Environmental Health Sciences Center, University of Rochester, Rochester, N.Y., for providing biological samples and atomic absorption data.
P. Mushak, F. E. Tibbetts, P. Zarnegar, and G. 6. Fisher, J. Chromatogr.,
LITERATURE CITED (1) P. A. Krenkle, W. D. Burrows, and R. S. Reimers, Crit. Rev. Environ. Control, 3,303-362 (1973). (2)J. F. Uthe and F. A. J. Armstrong, Toxicol. Environ. Chem. Rev., 2,45-77 (1974). (3)L. Fishbein, Chromatogr. Rev., 13,83-162 (1970). (4)P. Mushak, Environ. Health Perspect., 4,55-60 (1973). -(5) G. Westoo, Acta Chem. Scand., 22,2277-2280 (1968). (6)J. F. Uthe, J. Solomon, and 6. Grift, J. Assoc. Off. Anal. Chem., 55,585-589 (1972). (7)L. R. Kamps and 6. McMahon. J. Assoc. Off. Anal. Chem., 55,590-595, (1972). (8)M. L. Schafer, U. Rhea, J. T. Peeler, C. H.Hamilton, and J. E. Campbell, J. Aarlc. FoodChem., 23, 1079-1083 (1975). (9)J. E.congbottom, R. C. Dressman, and J. J. Lichtenberg,J. Assoc. Off. Anal. Chem., 56, 1297-1303 (1973). (IO)R. T. Ross and J. G. Gonzalez, Bull. Environ. Contam. Toxlcol., I O , 187-192 (1973) - -, (11) T GiovanoliJakubczak,M R Greenwood, J. C. Smith, and T. W. Ciarkson. Clin. Chem. ( Winston-Salem, N.C.), 20, 222-229 (1974). (12)R. Von Burg, F. Farris, and J. C. Smith, J. Chromatogr., 97, 65-70 (1974). I
( 1972).
87,215-226(1973). P. Jones and G. Nickless, J. Chromatogr., 76, 285-290 (1973). P. Zarnegar and P. Mushak, Anal. Chim. Acta, 69,389-407 (1974). P. Jones and G. Nickless, J. Chromatogr., 89,207-208 (1974). G. Schwarzenbach and M. Schellenberg, Helv. Chim. Acta, 48, 29-46
(1965). R . 6. Simpson, J, Am. Chem. Soc., 83,4711-4717 (1961). J. A. Ealy, W. D. Shults. and J. A. Dean, Anal. Chlm. Acta, 64,235-241 (1973). Y. Talmi and R. E. Mesmer, Water Res., 9,547-552 (1975). M. H. Abraham and G. F. Johnston, J. Chem. SOC.A, 1970,188-197. J. F. Thompson, A. C. Walker, and R. F. Moseman, J. Assoc. Off. Anal. Chem., 52, 1263-1277 (1969). R. Hartung, in "Environmental Mercury Contamination," R. Hartung and 6. D. Dimran, Ed., Ann Arbor Science Publishers,Ann Arbor, Mich., 1972, pp 157-161.
RECEIVEDfor review October 7, 1976. Accepted December 9, 1976. This research was supported by the Food and Drug Administration (Contract No. 223-74-2152) and grants from the National Institute of Environmental Health Sciences (ES-01247, ES-01248). This paper was presented at the 7th Northeast Regional Meeting of the American Chemical Society, Albany, N.Y., August 1976.
Determination of Tranexamic Acid in Biological Material by Electron Capture Gas Chromatography after Direct Derivatization in an Aqueous Medium Jorgen Vessman*
and Signhild Stromberg
AB KABI, Research Department, Analytical Chemistry, S- 1 12 8F Stockholm, Sweden
It acts as a competitive inhibitor on plasminogen, thereby decreasing the fibrinolytic activity. Blood levels have been measured by a method comprising ion exchange, high voltage electrophoresis, and colorimetry ( 2 ) .This method can measure therapeutic concentrations down to a few pg/mL, but is somewhat tedious. An alternative method with a higher capacity (20-30 samples a day) and even higher sensitivity was needed in a series of biopharmaceutical and toxicological studies. Numerous papers have dealt with gas chromatography of amino acids as derivatives ( 3 ) .Few have, however, used electron capture detection. The method of Husek comprising a single step derivatization with 1,3-dichlorotetrafluoroacetone can only be used for a-amino acids ( 4 ) .As early as 1963, Lipsky and Landowne demonstrated the excellent sensitivity of the 2,4-dinitrophenyl derivatives of the common amino acids ( 5 ) .This type of amino group derivative is high-boiling and this seems to have restricted its use mainly to low-boiling amines (6). Tranexamic acid (trans-4-aminomethylcyclohexanecarCrosby and Bowers reported on two reagents, alternative boxylic acid), Cyklokapron, is a synthetic w-amino acid with to 2,4-dinitrofluorobenzene, containing a trifluoromethyl useful antifibrinolytic properties ( I ) . group as well (?), which gave more volatile derivatives but with the same high sensitivity in the electron capture detector. CHZKHZ This paper describes a method based on electron capture gas chromatography of the esterified N-(2'-nitro-4'-trifluoromethylphenyl) derivative of tranexamic acid. HOOC The derivative has excellent gas chromatographic properties and is easily prepared in the aqueous phase at the ambient Tranexamic acid, AMCA temperature. The conditions for the formation of the derivative, its sensitivity and stability are discussed in the paper as well as some applications to human plasma samples. A Present address, AB Hassle, S-431 20 Mdlndal, Sweden.
Tranexamic acid is a synthetic amino acid wlth antifibrinolytic properties. A rapid gas chromatographic method has been developed for determinations in small biologicalsamples. The amino acids in the sample (5200 pL of plasma) are coupled in the aqueous phase directly to 4-fluoro-3-nitrobenzotrifluoride in a mixture of dimethylsulfoxide and borate buffer, pH 9.4. The acidic derivatlve is alkylated in an extractive alkylation step with tetrabutylammonium as the counterion. Quantitatlon is by electron capture gas chromatography. Both derivatlzation steps are performed in the presence of the biological sample and at room temperature and require only 5 min each. Samples of 250 ng/mL (50 ng altogether) can be analyzed with a relative standard deviation of f7%. A thin-layer chromatographic step is needed for samples below 1 pg/mL. The derlvatlve has an excellent electron capture response; 40 pg can be quantified in an injected sample (retention time 5 mln).
H
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preliminary communication on this work has been released (8).After completion of this study, a mass fragmentographic method for the determination of tranexamic acid was published by Miyazaki et al. (9). Tranexamic acid has been used as a most suitable internal standard by Gehrke et al. in the gas chromatographic analysis of the common a-amino acids (10).
EXPERIMENTAL Apparatus. A Varian 1400 gas chromatograph was used with a scandium 3H electron capture detector at 270 OC. The 1.5m X 1.8mm glass column was filled with a packing of 0.5%cyclohexanedimethanol succinate (CHDMS) or 1%OV-225 on Chromosorb G, acid-washed and silanized, conditioned at 250 "C overnight, or at 275 O F for 2 h. The column temperature was 250 "C and that of the injector 250 "C. The nitrogen flow rate was 45 mL/min. The electrometer sensitivity setting was usually X A/mV. All extractions were carried out in centrifuge tubes placed in a horizontal position in a mechanical shaker (60 strokedmin). Centrifugation was done in a Sorvall centrifuge GLC-2 at 2000 rpm. Reagents. Reagents used were dimethylsulfoxide (DMSO), and methylene chloride, both reagent grade, from Fisher, Fair Lawn, N.J.; a,a,a,-4-tetrafluoro-3-nitrotoluene obtained under the name of 4fluoro-3-nitrobenzotrifluoride (FNBT) from Pierce, Rockford, Ill.; toluene ana ethyl iodide, both reagent grade, from Merck, Darmstadt, German Federal Republic; tetrabutylammonium hydrogen sulfate (TBAHS) obtained from AB Biotec, Stockholm, Sweden; and sodium borate buffer (NaZB407),pH 9.4,0.2 M (saturated). Tetrabutylammonium Hydrogen Sulfate, 0.15 M,p H 9.5, was prepared by dissolving 51 g of tetrabutylhydrogen sulfate in 500 mL of water, adjusting pH to 9.5 with 0.1 M sodium hydroxide, and diluting with water to 1 L. Wash with 3 X 50 mL of methylene chloride. Ethyl Iodide 3 M in Methylene Chloride was prepared by diluting 25 mL of ethyl iodide with methylene chloride to 100 mL. When stored in darkness, it is stable for about a week. 4-Fluoro-3-nitrobenzotrifluoride reagent (0.03 M ) was prepared by diluting 0.4 mL FNBT to 100 mL with dimethylsulfoxide. Standards used were tranexamic acid (trans-4-aminomethylcyclohexanecarboxylicacid, AMCA) (contains 99% in the aqueous phase), the pH had to be >9.3 for toluene and equal phase volumes. With the volume ratio used (toluene-water, pH 9.5, 3:1), no extraction of nanogram amounts of the AMCA derivative into toluene occurred. Extractive Alkylation o f t h e N T P Derivative. The N T P derivative of AMCA had to be esterified before the gas chromatographic step and the most convenient method was the extractive alkylation technique (16). This is based on the extraction of the acid anion as an ion pair with a suitable quaternary ammonium ion into an organic solvent where alkylation occurs. In the studies of the extractive alkylation reaction, an acid washing step (0.1 M HzS04 5 mL) was included between points 8 and 9 in the procedure. This stopped further reactions in the organic layer, as the acid in nonprotolyzed form is considerably less reactive. Counterion. TBAH and tetrahexylammonium hydroxide (THAH) were compared with respect to their ability to serve as counterions in an extractive alkylation. As discussed by Ehrsson (17), THA gave a more rapid reaction than TBA. However, this could not be utilized as THA resulted in broad solvent peaks at the concentrations necessary for the analysis of serum samples. TBA was therefore used as a counterion although the reaction was considerably slower. ANALYTICAL CHEMISTRY, VOL. 49, NO. 3, MARCH 1977
371
AMCA
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Figure 5. Tranexamic acid in plasma after administration of 1 g iv TBAH Concentration. With an extraction time of 60 min, the same yield ratio was obtained with 0.003 to 0.30 M TBAH. With biological samples containing other amino acids, a high concentration (0.15 M) was necessary. pH. The yields were constant above pH 9. pH values above 11 gave rise to more disturbances than those below. The reaction rates at pH 9 and 13 were found to be the same. The Concentration of ethyl iodide was varied from 0.01 M to 3 M. The yield was quantitative for 3 M after 60 min; 1M required double the time (Figure 2). The rate limiting factor in the present aIkylation procedure is thus the amount of the alkylating agent. Because of a disturbing peak in the chromatogram, alkylation was carried out with ethyl iodide instead of methyl iodide. No difference in their reactivity was observed. Time Dependence with Acid Treatment. The time for quantitative alkylation was studied for AMCA and the internal standard. It was found that the reaction was complete after 60 min (Figure 2). The reaction was more rapid when DMSO was not present (-100% after 30 rnin). This retarding effect of the presence of DMSO most probably had to do with the fact that DMSO was partially extracted into methylene chloride (-10%). Time Dependence without Acid Treatment. The time dependence of the alkylation of NTP-AMCA was first studied 372
ANALYTICAL CHEMISTRY, VOL. 49, NO. 3, MARCH 1977
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Flgure 6. Tranexamic acid in plasma after oral administration.(0)0.5 g dose. ( 0 )2 g dose. under conditions in which the alkylation reaction was stopped by shaking the mixture with an acidic aqueous phase. This step is not included in the procedure as it was found possible to speed up the derivatization considerably during the evaporation step without previous acid treatment. Reaction mixtures which were not acidified but evaporated and reconstituted in toluene gave upon injection the same peak height ratio after 60 min as after 5 min. This further simplified the method. The reason for this increase in reaction rate is probably the increasing concentration of the alkylating agent during the evaporation procedure and also the higher temperature in the final stage. Internal Standard. MK-975 was chosen as the internal standard as the behavior in all critical respects was comparable to AMCA. The cis isomer of AMCA could be used but was rejected as it had a shorter retention time closer to the natural amino acids. Properties of the Derivatives. The stability of the esterified N T P derivative of AMCA was excellent. No decomposition occurred when the derivative in methylene chloride was shaken for 2 h either with 0.1 M hydrochloric acid or 0.1 M sodium hydroxide. The isomerization into the cis form, as recently reported by Miyazaki et al. (9) in their amidation step with heptafluorobutyric acid anhydride, was not observed with the present mild reaction conditions (derivative preparation at room temperature). The phenol resulting from the hydrolysis of the FNBT reagent gave after alkylation a product that could be extracted into toluene. This resulted in large solvent fronts. However, if the toluene layer was shaken with aq aqueous phase with a pH above 13,the product was redistributed to the aqueous phase and the influence of the by-product from the reagent was eliminated. The non-esterified NTP-AMCA derivative seemed, however, to be sensitive to hydrochloric acid 0.1 M and sodium hydroxide 0.1 M. However, NTP-AMCA will not stay without FNBT in an alkaline medium for more than a couple of minutes during washing with toluene. The gas chromatographic properties of the derivatives were excellent. Polar phases such as Carbowax terephthalic acid, OV-225 or cyclohexanedimethanol succinate were preferred. On these columns the AMCA derivative separated from the solvent front and some disturbing peaks. A typical chromatogram is shown in Figure 3. Samples of biological origin produced more peaks than aqueous ones, but no blanks were present. The phthalates, which so often disturb electron capture gas chromatograms, did not interfere with the derivatives. The retention of bis(ethylhexy1) phthalate on an
OV-225 column was 0.25 compared with the ethyl ester of NTP-AMCA. The detectability of the derivative in the electron capture detector was excellent. The minimum detectable quantity was about 40 pg with a retention time of 5 min, recorded with an OV-225 column at 235 "C. There was no temperature dependence of the detector response between 250 and 350 "C in a 63Nidetector. Behavior of Natural Amino Acids. The reaction of some natural amino acids with F N B T under the conditioris worked out for AMCA was studied briefly. It was found that all amino acids which contained functional groups in addition to one a-amino group and one carboxylic group (e.g., serine, glutamic acid, lysine, etc.) yielded derivatives which were too nonvolatile to be eluted under the gas chromatographic conditions chosen. The simple a-amino acids (glycine to leucine) all gave derivatives which were eluted at considerably lower temperatures than AMCA and t-aminocaproic acid (EACA), both w-amino acids (See Table I). Apparently, this structural difference influenced the retention behavior considerably'. This feature is an advantage when analyzing AMCA in the presence of large amounts of the natural amino acids. Isolation of AMCA from Biological sample!^ In the analysis of natural amino acids by gas chromatography in biological samples such as plasma, the first step is often deproteinization (18,19). The amino acids are then taken up on an ion exchanger, eluted and concentrated before >;hederivatization steps. Pellizzari et al. found that the individual recoveries after the purification steps varied considerably in a picrate denaturation method (20). After therapeutic doses, tranexamic acid products serum levels (1-15 pg/mL) which are in the same range as those of the natural amino acids (20,21). No attempts were made in the present study to dep roteinize samples containing proteins. Initially the amino acids were isolated on a cation exchanger in H+ form, both on column and batchwise. With I4C-labeled AMCA it was noticed that losses due to adsorption to the polystyrene resin occurred, ei3pecially for amounts below about 500 ng. It was possible to use the direct derivatization procedure for up to about 200 pL of plasma or serum and it caused no losses of AMCA and produced reproducible results with serum, plasma, and urine samples. Plasma and serum samples gave the same result. Tissue samples (e.g., muscle, )urethra, tumor) were first hydrolyzed in 6 N HCl. After pH adjustment, aliquots were then analyzed as serum samples. Recovery and Precision. The analytical procedure is outlined in the block diagram in Figure 4. By this method 30 samples could be analyzed per day. With therapeutic levels (>lpg/mL) an automatic injection system could be used (Hewlett-Packard 5710 7671) with an output of about 50 samples per day. Analysis of one sample from a biopharmaceutical study gave 11.96 f 0.17 Kg/mL with a relative standard deviation of 1.4% ( n = 8). Samples of lower concentration (235 ng/mL) required a cleanup, and the relative standard deviation was f ' 7 % ( n = 8). The relative recoveries were always around 100%. Exper-
+
iments with 14C AMCA, added to serum, produced absolute recoveries of around 60% at 250 ng/mL, i.e., 50 ng taken for analysis. This low value can partly be explained by quenching effects of the N T P derivative in the scintillation measurements and also losses in the extra handling steps required in these experiments. As the peak heights obtained from AMCA in plain aqueous solution were the same as those obtained from corresponding amounts in serum samples, the absolute recoveries can be assumed to be much higher than the figure given above. Applications to Biological Samples. The method has been used for more than two years for the determination of tranexamic acid in biological samples of various types. Examples demonstrating the usefulness of the procedure in the analysis of plasma samples are given in Figures 5, and 6, in which the time course of tranexamic acid concentration after oral and intravenous administration is shown. Samples taken later than 12 h after administration were usually so low in concentration (
