Gas-Chromatographic Determination of Inorganic Mercury and Organomercurials in Biological Materials Chris J. Cappon and J. Crispln Smith" Environmental Health Sciences Center, and Department of Pharmacology and Toxicology, University of Rochester School of Medicine and Dentistry, Rochester, N. Y. 14642
A method for the extraction, cleanup, and gas-chromatographic determination of organic (alkyl- and aryl-) and lnorganic mercury in biological materials has been developed. Methyl-, ethyl-, and phenylmercury are first extracted as the chloride derivatlves. Inorganic mercury is then isolated as methylmercury upon reaction with tetramethyltin. The Initial extracts are subjected to thiosulfate cleanup, and the organomercury species are isolated as the bromide derivatives. Total mercury recovery ranges between 75 and 90% for both forms of mercury, and is assessed by using appropriate 203Hg-labeledcompounds for liquid scintillationspectrometric assay. Specific gas-chromatographic conditions allow detection of mercury concentrations of l ppb or lower. Mean deviation and relative accuracy average 3.2 and 2.2%, respectlvely. The method provides good agreement between gas-chromatographic and atomlc absorptlon spectrometric data. On a routine basis, a skilled analyst can handle up to 24 samples dally.
There is a great demand for rapid and sensitive analytical methods for determining mercury in biological samples. T h e widespread environmental hazard a n d toxicological concern for mercury have stimulated research into various new methods of microanalysis (1, 2 ) . Gas chromatography employing electron-capture detection has been used quite extensively as a sensitive technique for analyzing organomercury compounds in biological materials ( 3 , 4 ) . Westoo ( 5 ) developed t h e first basic procedure for determining methylmercury and adapted i t t o various materials. Since then, several specific variations of this basic procedure have appeared for methylmercury analysis in fish (6-8), sediment ( 9 ) ,urine (IO), hair ( I I ) , and blood (12). All consist of an initial extraction of methylmercury as a halide with organic solvent, followed by cleanup prior t o gas-chromatographic analysis. There is also t h e necessity to quantitatively identify mercury in both its organic a n d inorganic forms in various biological media. Cold-vapor atomic absorption spectrometry (13) is commonly used for direct determination of total and inorganic mercury, organic mercury being reported as t h e difference between the total and inorganic results. However, this method does not permit determination of the specific form of organic mercury. A few gas-chromatographic procedures for measuring inorganic and organic mercury exist. Inorganic mercury is converted into an organomercurial by reaction in t h e sample media with arenesulfinates (14, 15), organometallic reagents (16), and trimethylsilyl derivatives (17). Not all of these procedures are routinely applicable to all sample types. Most require extensive extraction and cleanup steps, a n d employ a variety .of instrumental conditions. A simplified, rapid, and routine method for all sample types is highly desirable. In this paper, an analytical procedure is described for different mercury species in a variety of media. T h e procedure
is general for organic (alkyl- and aryl-) and inorganic mercury. Media studied with satisfactory results include: blood, grain, feces, fish, hair, milk, sediment, soft tissue, urine, and water.
EXPERIMENTAL Materials and Apparatus. A Brinkmann Polytron Model 3675 homogenizer LKinematica) is used for preparing aqueous homogenates of samples. Fifty-milliliter Nalgene polypropylene Oak Ridge centrifuge tubes (Nalge) are used throughout the extraction and cleanup steps. Sample extracts for gas-chromatographic (GC) and liquid scintillation spectrometric (LSS) assay are collected in 20-mL glass scintillation counting vials (Packard Instrument Co., Inc.). Reagents. All chemicals are of Analytical Reagent grade and distilled water is used for reagent preparation. Cysteine.HC1 was obtained from Sigma Chemical Company. Tetramethyltin (99% pure) was obtained from Aldrich Chemical Company and the methanolic reagent is prepared fresh prior to analysis. Benzene (thiophene-free and spectro-grade) was obtained from Mallinckrodt Chemicals. All reagents, except tetramethyltin, are extracted with benzene to remove any potential interfering GC contaminants. After preparation, 500 mL of reagent is extracted three times with 50 mL of thiophene-free benzene. Florisil (Florindin Co.) was 60/100 mesh in size. All reagents and solvents, with the exception of methanolic tetramethyltin, are stored in, and dispensed from, amber 1-L Oxford pipettors (Oxford Laboratories), with provisions for dispensing up to 10 mL of reagent. Standards. Inorganic and organomercury fortification solutions are prepared from the chloride salts obtained from K and K Laboand stored at ratories. HgC12 solutions are prepared in 0.5 N "03 0 "C in amber glass bottles. Methyl-, ethyl-, and phenylmercuric chloride are dissolved in 0.05 N NaZC03. The organomercury bromide salts used for preparing GC standards were purchased from the same source. Stock solutions (1 ppm RHgBr) are prepared in spectrograde benzene and appropriate dilutions are made to prepare working standards ranging between 0.05 and 2.0 ng/lO pL. Working standards are prepared fresh weekly in glass scintillation vials and stored in the dark t o prevent photodecomposition of RHg+. 203HgC12in 0.5 N HNO:j and CH:jZo3HgC1 in 0.02 N Na2C03 were obtained from New England Nuclear. C2H,j20:3HgCI and CeH5203HgOA~ were obtained in solid form from Amersham-Searle Corporation and must be dissolved in 0.02 N NaZC03. All isotopes are usually >99?? in radiometric purity, and the range of specific activity is 0.5-3.0 mCi/mg Hg. Appropriate dilution of the stock solutions is made to prepare working (spike) solutions containing approximately 6000 dpm and less than 1 ng Hg/lO kL. All isotope solutions are stored at 0 "C inside lead containers. 2'J'HgLSS standards are prepared fresh and in triplicate for each analysis. An amount of isotope solution equal to that used to spike the sample prior to extraction (usually 10 pL) is placed in a scintillation counting vial and 20 mL of Aquasol Universal LSC Cocktail (New England Nuclear) is added. Sample Preparation. Whole samples, aqueous homogenates, or alkaline digests of samples can be used for analysis. Table I lists biological materials that lend themselves to the three methods of sample preparation. Aqueous Homogenates. A 0.5-2.0 g portion of finely-chopped sample and an equal volume of distilled water are homogenized to a uniform consistency with the Polytron homogenizer and diluted to a known final volume (usually 10 mL) with water. Alkaline Digests. A 0.5-2.0 g portion of finely-chopped sample is placed in a 20-mL glass vial. Two mL of 45% NaOH and 1 mL of 1% cysteine-HC1 are added. The contents are gently heated until the sample has dissolved. Boiling of the mixture must be avoided. The mixture is allowed to cool and diluted to a known final volume (usually 10 mL) with 1%NaCl. ANALYTICAL CHEMISTRY, VOL. 49, NO. 3, MARCH 1977
365
Sample ( Pratein-bound RHg+and Hg?
Table I. Sample Preparation Preparation Whole sample Aqueous homogenate Alkaline digest
Aqueous ( L i b a r a ~ a d~
I 203HgC12, 0 2 M mnhanalic Meq%
I R z o 3 H g C I , 8 M Urea,
Samples
0 5M CuS04, 3N HCI
Blood, grain, sediment, urine, water Feces, fish, sediment, soft tissue Blood, feces, fish, grain, hair, soft tissue
10 min
2 Benzene IOmin .5min
Benzene [ RHpCI)
100 %
$ 7
2 Benzene 10min Benzene (MeHgCI)
85-90%
AqueDUs ( M e H i )
100 %
0 01 M Na2S203 15 sec
Table 11. Gas Chromatography Column: Coiled glass, 1.22-m length, 4-mm i.d. Packing: 1.5%, OV-17 1.95%, QF-1 on SO/lOO Chromosorb W-HP (Alltech Associates) Iklstrument settings: Temperature, DC: Inlet Column Detector 130 110 150 MeHg, EtHg PhHg 185 180 185
+
Carrier flow rate: 120 cm3/min, nitrogen Detector settings: A full-scale (AFS) Sensitivity: 3 X Suppression current: 1-2 X 10-7 A Potential: 5 V Retention times, min MeHgBr: 0.6 EtHgBr: 1.6
100%
I
I I 0 5 M Cu8rg
2 Benzene. 3 0 m 3 I I Na,SO,-
I, (RHgBr) Bsnzene
,
Florisil
80- 90 K GC,LSS
1
Bsyrm (MeHgBrl overall recovery
80-90 %
.
75- 90 %
Flgure 1. Outline of HCI-Me&n procedure
PhHgBr: 2.0
These preparation procedures are only general. Since the original sample is diluted in each case, the optimum amount of sample and dilution will be dictated by the level of mercury present in the sample and the instrumental sensitivity and detection limits of the method. An example illustrating the effect of sample preparation on mercury detection is presented elsewhere in this paper. Unprocessed grain samples are usually analyzed whole since most of the mercury is present on the surface from fungicidal treatment. Since processed grain is impregnated with mercury, alkaline digests can be used. Sediment samples do not digest completely and must be analyzed whole or as homogenates. Procedure. The sample (0.5-2.0 g, mL) is placed in an Oak Ridge tube. A 10+L aliquot of the appropriate RZa3HgC1spike is added by an Eppendorf pipette, followed by 2 mL of 8 M urea. The spiked sample is mixed 10 s on a Vortex Genie mixer (Scientific Products), and allowed to stand 10 min to provide sufficient time for z03Hgto bind to the sample matrix sites. One mL of 0.5 M CuSO4, and 3 mL of 3 N HC1 are added. The contents are mixed and let stand 5 min. Ten mL of thiophene-free benzene is added, and the mixture is shaken 10 rnin on a Thomas-Borner shaking apparatus (Arthur H. Thomas Co.) and centrifuged 5 min at 3000 X g in an IEC Model C centrifuge (International Equipment Co.). Samples that emulsify (especially whole blood and homogenates of fish, feces, and tissue) are centrifuged 10 min at 15 000 X g in a Sorvall Model RC-5 superspeed refrigerated centrifuge (Dupont Co., Instrument Div.). The benzene layer is removed and placed in a second Oak Ridge tube. Five mL of thiophene-free benzene is added to the aqueous layer, and the contents are shaken 5 min and centrifuged as before. The benzene layers are combined and the aqueous layer is kept for inorganic mercury extraction. One mL of 0.01 N NazSz03 is added to the combined benzene layers, and the contents are mixed vigorously 15s on the Vortex mixer and centrifuged 5 min a t 3000 X g. The benzene layer is discarded. If the aqueous layer is emulsified, 1 mL of 95%ethanol is added at this point. To the aqueous layer 0.5 mL of 0.5 M CuBrz is added, and the contents are mixed 10 s. An appropriate volume of spectrograde benzene (usually 1-2 mL) is added, the mixture is shaken 30 s and c e n t r i f u p m i n at 3000 X g, and the benzene is transferred to a glass test tu e containing approximately 0.1 g of 1:1 (v/v) anhydrous NazSO4-Florisil. After mixing 5 s, the benzene is transferred to a glass scintillation vial and submitted for GC analysis. The aqueous layer from the initial benzene extractions is spiked with 10 ~ LofL 203HgC12solution, mixed 10 s, and allowed to stand 10 min for z03Hgincubation. Methanolic 0,2 M tetramethyltin (0.5 mL) is added, and the mixture is shaken 10 rnin on the Thomas shaker. Ten mL of thiophene-free benzene is added. Shaking is continued 10 min and the mixture is centrifuged as described above. The benzene layer is transferred to a second Oak Ridge tube and subjected to the identical cleanup and isolation steps described for the organic mercury extraction scheme. 366
A q u e w s (RHO')
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If necessary, the procedure can be interrupted after completion of the thiosulfate cleanup step in both the organic and inorganic mercury extractions. After the benzenalayer is discarded, the aqueous thiosulfate layer is refrigerated overnight. Determination of inorganic mercury and organomercurials present in a sample involves the use of sequential or simultaneous analysis depending on the form of the organomercurial. If methylmercury is present, the sequential analysis scheme described in this section is performed, resulting in two extracts for GC and LSS analysis. When either ethyl- or phenylmercury is present, both the inorganic and the organic forms can be determined simultaneously, shortening the overall procedure. In this case, 203HgC1zspike and tetramethyltin reagent are added to the sample along with RZo3HgCl,urea, CuSO4, and HCl. The contents are shaken 10 min. Ten mL of benzene is added, and the procedure is continued as described previously for organomercury extraction. Samples containing only inorganic mercury are analyzed by the same procedure, the RZo3HgC1spike being deleted. While simultaneous extraction can also be done for phenylmercury, GC temperature programming would be necessary to avoid excessive retention time for phenylmercury?although such a situation has not been encountered in this laboratory. Oak Ridge tubes can be used indefinitely. However, because of high instrumental sensitivity, traces of mercury contamination must be removed from the tubes upon Completion of each analysis. The tubes are soaked in a sulfuric acid solution of Chromerge Labware Cleaner (Monostat Corp.), followed by soaking in Lift-Away decontaminant solution (Research Products International Corp.) to remove Po:IHg contamination. Gas Chromatography. A Packard Model 7401 gas chromatograph (Packard Instrument Co., Inc.) equipped with a Model 810 electron-capture detector is used. The detector employs a 150 mCi :'H foil and is operated in the dc mode. The instrument operating conditions are presented in Table 11, along with corresponding retention times for methyl-, ethyl-, and phenylmercuric bromide. Syringes are cleaned weekly by soaking the barrel and stainlesssteel plunger in syringe cleaning solution (Regis Chemical Co.). The silanized glass wool (Applied Science Laboratories) of the column inlet and outlet ends is replaced on a weekly basis. The detector cell and probe are periodically cleaned by soaking 5 min in a warm solution of 4:2:1 water-sulfuric acid-nitric acid. The parts are then washed thoroughly with distilled water, rinsed with acetone, and dried overnight in an oven. The "H foil is cleaned by soaking in 5% methanolic KOH and rinsing with methanol. Liquid Scintillation Spectrometry. A Packard Model 2450 Liquid Scintillation Spectrometer System, equipped with a ServoTray Program Control Model 2409, is used to measure zO:iHgbetaactivity in sample extracts. Upon completion of GC analysis, the sample is diluted to 20 mL with Aquasol Universal LSC Cocktail and counted 10 min in the spectrometer along with the appropriate zO:iHg spike standards.
RESULTS AND DISCUSSION Sample Preparation. Alkaline digestion was first developed exclusively for hair ( 11 ) and is especially advantageous
Table 111. Analytical a n d Recovery Data Added MeHgCla (ng/g)
Mean (ng/g)
% Mean deviation
% Relative
% Recovery
Sample
accuracyc
(average)
Blood Feces Liver
139.2 12.7 196.1
141.4 12.4 203.6
1.3 4.9 2.8
1.6 2.4 3.8
79.8 86.3 85.0
Added HgClz" Blood 198.0 194.6 3.4 1.7 83.4 22.4 21.9 5.6 2.2 88.2 Feces Liver 192.7 190.1 1.3 1.3 81.5 Whole sample. All samples were analyzed as alkaline digests. Mean values are the average of three analyses. All have been corrected for recovery. c Percent relative accuracy of the mean.
for blood, fish, feces, and soft tissue. Sodium hydroxide digests are homogeneous, provide more uniform sample distribution, and are not prone to emulsification during the initial acid extraction as are aqueous homogenates. Consequently, high-speed centrifugation is not necessary. In addition, mercury recovery is more efficient due to breakdown of protein and lipid material in the sample matrix during digestion. Total mercury recovery is usually 5-10% higher than that obtained with aqueous homogenates and whole samples. Final benzene extracts are cleaner. T h e use of cysteine-HC1 in the digestion procedure serves to complex the organic mercury and prevents breakdown to inorganic mercury. However, cysteine is gradually oxidized in the strong alkaline solution, and organic mercury slowly breaks down after a few days. Therefore, the sample must be analyzed soon after digestion. P r o c e d u r e . T h e procedure is schematically outlined in Figure 1. Urea aids in enhancing both 203Hgincubation and organic mercury recovery in the initial extraction step by uncoiling proteins in the sample matrix and exposing mercury-binding sulfhydryl sites for acid cleavage. Use of urea was first applied in this laboratory for methylmercury analysis in blood (12).T h e first benzene extraction results in 85-90% recovery of organic mercury. A second extraction removes any remaining organic mercury, and is necessary for samples containing inorganic and methylmercury, since the isolation and quantitation of inorganic mercury is based upon its conversion to methylmercury. Sodium thiosulfate, which has a high complexing affinity for organic mercury (18),provides rapid quantitative cleanup of the initial benzene extracts. Use of cupric bromide permits high mercury recovery while keeping the volume of organic solvent small and minimizing extraction time. T h e choice of bromide as the halide source is based upon its greater nucleophilic character and the more favorable distribution between the organic and the aqueous phase for RHgBr compounds (19,20).RHgBr derivatives are less subject t o photochemical decomposition than are the corresponding iodide derivatives (21).In addition, cupric ions allow more complete liberation of organic mercury from its thiosulfate complex. Organic mercury recovery for the final extraction step is 80-9OOh. Total recovery is 7 5 9 0 % and is somewhat dependent upon the actual sample matrix and preparation method. Abraham and Johnston (22)studied the ability of various peralkyltin compounds t o alkylate inorganic mercury to monoalkylmercury. Zarnegar and Mushak (16) attempted to measure inorganic mercury in various samples with tetramethyltin. Variable low yields of methylmercury resulted because of partial decomposition of tetramethyltin in the strongly acidic media employed. In contrast, extraction of inorganic mercury by the HCl-Me4Sn procedure is made using milder acidic conditions for methylation. Addition of methanolic tetramethyltin to the initial aqueous phase from
Table IV. GC-AA Intercomparison Study PPm Hg
GC
Sample
AA
GC/AA
as MeHg Fish Hair Muscle
1.10 266.2 0.72
1.06 272.9 0.70
1.04 0.98 1.03
as EtHg Blood Kidney
0.72 0.66
0.77 0.68
0.94 0.97
as inorganic Blood Fish Sediment
0.59 0.08 0.17
0.57 0.07 0.19
1.04 1.14 0.89
the organic mercury extraction effectively produces methylmercury in 85-90% yield after 10 min of shaking. T h e methylmercury is simultaneously converted to the chloride derivative before extraction into benzene and subsequent cleanup (Equation 1).
+
(CH3)4Sn Hg2+
%CH3HgCI + (CH3)3SnCl
(1)
The accuracy and precision of the method were evaluated by analyzing different sample types fortified with mercuric chloride and methylmercuric chloride (Table 111).Mean deviation and relative accuracy averaging 3.2 and 2.2%, respectively, were observed. In the past, most of our routine GC work was performed to verify atomic absorption (AA) data. Thus, it was also important t o examine the degree of GC-AA correlation obtainable with this procedure. Several samples previously analyzed by an AA procedure (13)were analyzed by GC. GC-AA comparison results for several representative samples are given in Table IV. There is good agreement between the two methods for samples containing methyl-, ethyl-, and inorganic mercury, and this is expressed in terms of GCIAA ratios. Since using the HCI-Me4Sn procedure for routine work, we have consistently achieved good correlation with AA data. G a s C h r o m a t o g r a p h y . Thompson e t al. (23) found the mixed phase packing of 1.5%OV-17 1.95% QF-1 very suitable in overall performance for pesticide analysis. Hartung (24) successfully applied 11%OV-17 QF-1on Gas-Chrom Q for methylmercury and dimethylmercury analysis. T h e packing 1.5%OV-17 1.95%QF-1 on Chromosorb W-HP was chosen for this work and has proved superior for routine use. This packing permits column operation a t a relatively lower
+ +
+
ANALYTICAL CHEMISTRY, VOL. 49, NO. 3, MARCH 1977
367
A
MeHgBr
"g*
, PhH;
Tim (min.) Time (min. 1
Figure 2. Chromatograms of RHgBr GC standards Concentration of each bromide is 0.5 ng/lO WLinjection. Recorder chart speed is 12 inches/h B
A
Figure 4. (A) Chromatogram of heart sample (alkaline digest) containing 1.32 ppm ethylmercury and 0.07 ppm inorganic mercury. (B) Chromatogram of egg yolk sample (aqueous homogenate)containing 0.63 ppm phenylmercury
HeHg*
0
1
2
3
Time ( min.)
Figure 3. Chromatograms of liver sample (alkaline digest) containing (A) 24.46 ppm methylmercury and (B) 17.86 ppm inorganic Hg
temperature (110 "C)for methyl- and ethylmercury. I t also provides excellent sample peak resolution a t very short retention times, minimizing column bleed, and extends column life without excessive loss of detector sensitivity. One such column has been used routinely for 5 months without apparent loss of efficiency. T h e same column packing is also used for phenylmercury, b u t a t a column temperature of 180 "C. The bromides of methyl-, ethyl-, and phenylmercury are used to prepare daily standard calibration curves which are expressed in terms of peak height vs. organomercury bromide concentration. Typical chromatograms are shown in Figure 2. Each peak represents 0.5 ng of organomercury bromide per 10 pL of benzene injection. The curves are linear up to a t least 2.0 ng/lO pL injection. Slight differences in detector response on a weight basis are noted for each of the three organic forms. For the given instrument settings, the minimum detectable organomercury bromide concentration is 0.02 ng/lO pL, or 2 p p b RHgBr. Expressed as mercury, this represents minimum detectable levels of 1.36,1.30, and 1.12 ppb for methyl-, ethyl-, and phenylmercury, respectively. For a 2-g sample (whole) containing methylmercury, assuming 1mL of final extract and 8Wo recovery, this translates to a sample concentration of 0.85 ppb Hg. If 2 g of a sample containing methylmercury is used to prepare 10 mL of aqueous homogenate or alkaline digest and a 1-mL aliquot is analyzed, assuming equal extract volume and recovery, the mercury concentration giving the same instrument response would be 8.5 ppb. The minimum detectable concentration can be further lowered by increasing detector sensitivity but a t the expense of increased baseline noise level. 368
ANALYTICAL CHEMISTRY, VOL. 49,
NO. 3, MARCH 1977
In general, benzene extracts from this procedure are quite clean, as seen in Figures 3 a n d 4. T h e organomercury peaks are well resolved from any contaminants, and any late peaks elute within 3 min from sample injection. No attempts were made to identify any contaminant peaks present on the chromatograms. However, such peaks may arise from other electron-capturing organic components present in and extracted from the original sample matrix (e.g., chlorinated pesticides from grain or P C B residues from fish) rather than from the reagents or the extraction solvent. GC analysis of the final benzene extracts from reagent purification revealed no interfering peaks in the RHgBr regions. Liquid Scintillation Spectrometry. This technique has proved valuable for assessing mercury recovery by the described procedure. Monitoring with 203Hg-labeledcompounds permits straightforward determination of stepwise and overall recovery. The GC analyses and calculations are simplified because the addition of mercury standards to samples is avoided. Since less t h a n 1 ng of Hg is added to the sample before extraction, no correction need be made for the added spike except for samples of very low mercury content. Analysis and Calculations. GC quantitation of samples is accomplished by measuring peak heights from 10-pL sample injections and obtaining the concentration from standard calibration curves prepared daily. Relevant equations illustrating the use of 203Hg recovery data for calculating the amount of mercury in a sample are: % Recovery RHgBr =
cpm in benzene extract cpm in 10 pL spike std
x 100 (2)
where cpm = counts per minute. ng RHgBr injected 10 pL injection 100OpL Y mL benzene extract X x 100 mL benzene extract % Recovery - ng RHgBr injected x 10" x Y (3) % Recovery
Total ng RHgBr in sample =
In Equation 3, Y represents the volume of final benzene extract, since the final extract may have to be diiuted for samples of high mercury content. In reporting results, RHgBr is conventionally expressed as Hg using Equation 4. p p b Hg in sample =
Total ng RHgBr in sample Sample weight (g) X
atomic weight Hg molecular weight RHgBr
(4)
ACKNOWLEDGMENT 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.
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
L. Magos and T. W. Ciarkson, J. Assoc. Off. Anal. Chem., 55, 966-971 ( 1972).
P. Mushak, F. E. Tibbetts, P. Zarnegar, and G. 6. Fisher, J. Chromatogr.,
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 a t 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
I t 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 a t the ambient Tranexamic acid, AMCA temperature. The conditions for the formation of the derivative, its sensitivity and stability are discussed in the paper Present address, AB Hassle, S-431 20 Mdlndal, Sweden. as well as some applications to human plasma samples. A
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
ANALYTICAL CHEMISTRY, VOL. 49, NO. 3, MARCH 1977
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