basis because of the possibility of variations in instrumental conditions as shown in an earlier study (21). For this purpose, multiple ion detection using an accelerating voltage alternator (AVA) (23) and simultaneous monitoring of several ions, including one or more of the isotopically labeled internal standard, is necessary. Ideally, the internal standard should be subjected to the identical sample preparation and derivatization conditions as the compound in question. This was not possible in this specific case, as the labeled analogue of R T 3 had to be prepared separately during esterification. This problem can be alleviated by using a compound containing deuterium atoms on the thyronine phenyl rings, which, of course, would be a major project in itself. Nevertheless, the methodology developed for the analysis of free RTB by Dialysis-GC-MS-SIM can be applied to the analysis of all the thyroid hormones and their precursors as their HFB-ME derivatives.
ACKNOWLEDGMENT The authors thank B. L. Karger and R. W. Giese for helpful suggestions and T. Hanai for the capillary GC column.
LITERATURE CITED ( 1 ) P. R. Larsen, Med. Clin. North Am., 59, 1063 (1975). (2) P. Nicod, A. Burger, V. Stacheli, and M. V. Vallotton, J . Clin. Endocrinol. Metab., 42, 823 (1976). (3) H. Meinhold, K. W. Wenzel, and P. Schurnbrand, 2. Klin. Chem. Klin. Biochem., 13, 571 (1975). (4) I. J. Chropra and B. F. Crandell, N . Engl. J. Med., 293, 740 (1975).
(5) D. A. Fisher, N. Engl. J. Med., 293, 770 (1975). (6) K. A. Sterling and A. Hegedus, J . Clin. Invest., 41, 1031 (1962). (7) S. S. Levinson and S. U. Reider, Clin. Chem. ( Winston-Salem, N.C.), 20, 1568 (1974). (8) J. S. Annlno and R. W. Glese, “Clinical Chemlstry”, Llttle, Brown & Co., Boston, Mass., 1976, p 306. (9) K. A. Sterllng and M. A. Brenner, J. Clin. Invest., 45, 153 (1966). (IO) M. Tajuddin and S. G. Elfbaum, Clln. Chem., ( Winston-Salem, N.C.), 19, 109 (1972). (1 1) N. N. Nihei, M. C. Gershengorn, T. Mltsuma, L. R. Stringham, A. Cordy, B. Kuchmy, and C. S. Hollander, Anal. Blochem., 43, 433 (1971). (12) P. I. Jaakonmaki and J. E. Stouffer, J. Gas Chromatogr.,5 , 303 (1967). (13) J. E. Stouffer, P. I. Jaakonmaki, and T. J. Wenger, Blochim. Biophys. Acta, 127, 261 (1966). (14) B. A. Petersen, R. N. Hanson, R. W. Giese, and B. L. Karger, J . Chromatogr., 126, 503 (1976). (15) A. Dibbo, J. C. P. Sly, L. Stephenson, T. Walker, W. K. Warburton, and K. D. E. Whiting, J. Am. Chem. Soc., 90, 2690 (1968). (16) K. M. Baker, M. S. Shaw, and D. H. Wllliams, Chem. Commun., 1108 (1969). (17) E. Gelpi, W. A. Koenig, J. Biibert, and J. Oro, J . Chromatogr. Scl.. 7, 604 (1969). (18) A. M. Lawson, D. B. Ramsden, P. J. Raw, and R. Hoffenberg, Blomed. Mass Spectrom., 1, 374 (1974). (19) M. M. Bursey, J . Am. Chem. Soc., 91, 1661 (1969). (20) M. M. Bursey and M. K. Hoffman, J . Am. Chem. Soc., 91, 5023 (1969). (21) D. J. Marshall, 6. A. Petersen and P. Vouros, submitted for publlcation. (22) B. A. Petersen, R. W. Glese, B. L. Karger, and P. R. Larsen submitted for aublicatlon. (23) b F . Holland, C. C. Sweely, R. E. Thrush, R. E. Teets, and M. A. Bieber, Anal. Chem., 45, 308 (1973).
RECEIVED for January 31,1977. Accepted May 9,1977. This work was supported by a grant from the National Institutes of Health, (GM 22787).
Determination of Mefruside and Its Metabolites in Urine by High Performance Liquid Chromatography C. J. Llttle,” A. D. Dale, D. A. Ord,’ and 1.R. Marten Roche Products Ltd., P.O. Box 8, Welwyn Garden City, Hertfordshire, United Kingdom
A method Is descrlbed for packing high performance llquld chromatography (HPLC) columns using isopropanol as the slurry medium. Hlghly reproducible columns can be made by this technlque which applies equally well to reversed phase columns. Chromatographlc condltlons are described sultable for the analysls of a urine extract of the sulfonamide “Mefruslde” and Its metabolttes. Prlor to extractlon wlth ethyl acetate, the urine should be acidified, to convert all of the major metabollte (50x0-Mefruslde) Into its lactone form. The extract is then quantltatively analyzed by an external standardization technlque whlch gives essentially the same result as a radiochemical check method.
Sulfonamide drugs have been important for many years as antibacterial agents. Recently they have been used as antidiabetic and diuretic agents. Of the many and varied methods described for their analysis, chromatography is one of the more specific. Paper and thin-layer chromatography have been used extensively ( I ) , as have chromatography on polyamide (Z), zinc ferrocyanide ( 3 ) ,and paper impregnated with strong cation-exchange resins ( 4 ) . Recently, high pressure Present address, Spectra-PhysicsLtd., 17 Brick Knoll Park, St. Albans, Hertfordshire, U.K.
anion exchange (5),reverse phase (6),and ion pair partition (7)methods have been published for specific sulfonamides. This paper describes the application of high performance liquid chromatography (HPLC) on silica columns to the analysis of the sulfonamide Mefruside “I” (Ro-8-3725) and its metabolites “11” and “111” (Figure 1).
EXPERIMENTAL Apparatus. Modular chromatographic equipment was used comprising the following items: (a) Constametric I1 pump, (b) Rheodyne 7010 injection valve, (c) Cecil CE 212 UV monitor (set at 248 nm). Column packing was performed with a high pressure pump (maximum -15000 psig) from Stansted Fluid Power, Stansted, Essex. “Apollo” stainless steel tubing, with a ‘/2-wm internal surface finish was used throughout (Accles and Pollock, Oldbury, Warley, Worcestershire, U.K.). Reagents. All solvents were of analytical reagent (AR) grade and were used without further purification. Microparticulate silica used in the course of this work was Partisil-5, and was used straight from the bottle. TLC investigations were carried out on precoated plates (E.M. Merck, Darmstadt-BDH) which were used without activation. Samples of Mefruside “I” and its metabolites “11” and “111” were obtained from Bayer. Column Preparation. The balanced density slurry method of packing columns (8-10) and several variants of the method ( 1 2 , 12) are now common practice. For several years we have slurried silica and bonded silicas in alcohol mixtures which rapidly wet the silica surface. Initially we used methanol but now use isoANALYTICAL CHEMISTRY, VOL. 49, NO. 9, AUGUST 1977
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I ,009,
l
I
”
”
1
1
2
3
1
2
i
f l o w rate Irnl/minl
111
5 - 0 x o - liel’Yslde
lr(a-l1-91301C‘OC1
Figure 3. Flow dependence of column efflclency “H” (lower) and resolutlon “R” (upper). In the upper figure “R” is between the SUIfonamlde I and 111 and was calculated from /? = 2At/( W , W3). Conditions a s for Figure 2
+
Flgure 1. Chemical structures of Mefruside and two of its metabolites 2
(bi
-i 1
A
15
L 7, T 1
-
n5
?C
-
T
10
iminrl
-
0
Figure 2. Chromatogram of sulfonamides. Column, Lichroscfb SI 60-5 pm (50 cm), eluent, hexane -I-ethyl acetate (1:l);flow rate, 1.5 mL/mln propanol which produces a more stable slurry. The slurry was introduced into a column precolumn assembly via a long hypodermic needle. Methanol was then pumped through the column. All columns reported in this paper were packed by this method a t 7.5000 psig for 30 min. For convenience and economy the slurry composition was adjusted such that a minimum of support was left in the precolumn after the column has been packed. The reliability of this method can be judged from the fact that during 1976, some 70 columns (some of which were reversed phase columns) were packed in this manner, all of which were of a similar standard. We feel that this reliability of packing is in no small way attributable to the mirror finished “Apollo” tubing that was used. Further modification of the slurry can be achieved by the addition of a few percent of glycerol. Using this technique, even 40-km particles can be slurry packed with ease. Procedure. Examination of the sulfonamides 1-111 by TLC hexane (1:l) as eluent. suggested the use of ethyl acetate Transference of this procedure to the column was relatively easy, a typical chromatogram being illustrated in Figure 2. Optimization of flow rate was readily deduced from the flow dependence of the Height Equivalent to a Theoretical Plate (HETP) as shown in Figure 3. Obviously, maximum efficiency for all solutes in the mixture occurs at a flow rate of about 0.4 mL/min. In practice one has
2 0 15
10
5
10
5
J
0
0
Figure 4. Chromatogram of sulfonamides (trace levels). Column, as in Figure 2, eluent, hexane ethyl acetate (1:l); flow rate, 2 mL/rnin. Compounds separated are ( a )0.1 % I (peak 2) in I11 (peak 3)and ( b ) 0.1 % I11 (peak 3) In I (peak 2)
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ANALYTICAL CHEMISTRY, VOL. 49, NO. 9, AUGUST 1977
lminrl
8
7
6
5
L
3
2
1
0
Flgure 5. Chromatogram of urine extract. Column, Partisll 5 (25 cm); eluent, hexane -I-ethyl acetate (1:l); flow rate, 2 mL/min
for at least 30 min to allow for complete lactone formation. The urine was then extracted with ethyl acetate (3 X 10 mL) and the organic phase was blown to dryness under a stream of nitrogen. The residue was redissolved in ethyl acetate (3.0 mL) and analyzed by the method previously described. The chromatography of the biological extract is shown in Figure 5. The analysis was also performed radiochemically as a check procedure.
/
114
RESULTS
I
1
2
3
1
5
m g /mi
Figure 6. Calibration curves for Mefruside I and 5-oxo-Mefruside I11
to compromise between loss of efficiency and decrease in analysis time. It can be seen from the superimposed plot of Resolution “(R)” between I and I11 vs. flow rate (Figure 3a) that very little resolution is lost if the flow rate is increased beyond the optimum value, resulting in a desirable decrease in analysis time while still preserving adequate resolution. In biological systems there is often the need to detect traces of unchanged drug in the presence of large quantities of its metabolite and vice versa. The chromatographic system described is compatible with these requirements (Figure 4). To evaluate this methodology for real samples, a human volunteer was given an oral therapeutic dose of 14C-labeled Mefruside (18.9 pCi per 26 mg). Urine was collected from the volunteer and extracted in the following way. Urine (10 mL) was adjusted to pH 2 with dilute hydrochloric acid. This was necessary to convert any ring opened metabolite back to the lactone as in Figure 1. The acidified sample was left
Quantitation of the chromatography was achieved by an external calibration method. Linear calibration cwves (Figure 6) were achieved over the concentration range investigated (0-3 mg/mL). The results achieved by the chromatographic method showed the presence of 27 pg 5-oxo-Mefruside per 10 mL urine whereas the radiochemical method indicated 30 pg 5-oxoMefruside per 10 mL urine. This is very good agreement, particularly when it is realized that the radiochemical method is known to include about 10% of minor radioactive metabolites other than 5-oxo-Mefruside. No interference was observed from paracetamol, an increasingly common analgesic which often interferes with the analysis of biological extracts.
LITERATURE CITED (1) (2) (3) (4) (5) (6) (7)
(8) (9) (IO) (11) (12)
J. P. Comer and I. Comer, J . Pharm. Sci., 56, 413 (1967). Y-T. Lln, K-T. Wang, and T-I. Yang, J . Chromatogr., 20, 610 (1985). A. (3, Fogg and R. Wood, J . Chromatogr., 20, 613 (1965). D. J. Pletrzyk and E. Chan-Santos, J . Chromatogr., 67, 543 (1973). T. C. Kram, J . Phsrm. Sci,, 61, 254 (1972). L. D. Blghley and J. P. McDonnell, J . Pharm. Sci., 64, 1549 (1975). S. C. Su, A. V. Hartkopf, and 8. L. Karger, J . Chromatogr., 119, 523 (1976). R . E. Majors, Anal. Chem., 44, 1722 (1972). B. Coq, C. Gonnet, and J. L. Rocca, J . Chromatogr., 106, 249 (1975). P. J. N. Webber and E. H. McKerrell, J . Chromatogr., 122, 243 (1976). J. J. Kirkland, J . Chromatogr., 125, 231 (1976). H. R. Llnder, H. P. Keller, and R. W. Frel, J . Chromatogr. Sci., 17, 234 (1976).
RECEIVED for review September 27,1976. Accepted April 20, 1977.
Determination of Selenium in Blood Serum by Proton-Induced X-ray Emission Marina Bertl, Glampaolo Buso, Paolo Colauttl, Giullano Moschlnl, * Blanca Maria Stlevano, and Crlstlna Tregnaghl Laboratori Nazionali dell’ Isfituto Nazionale di Fisica Nucleare, Via Romea n. 4, 35020 Legnaro (Padova), Italy
Selenium determlnatlon In blood serum has been tested for thlck (dry-ashed) samples uslng x-ray emlsslon spectra Induced by 1.8- to 4-MeV proton beams. For quantltatlve analysls the Internal standard method has been used. Thls paper presents the procedure and evaluatlons of the sample preparation and analysls. The statlstlcal countlng error Is as low as 6 % and the sensltlvlty 1 1 0 ppb for 100-mln counts with 1.8-MeV protons and for 30-mln counts with 4-MeV protons.
A detailed review about the biological role of selenium has recently been carried out by R. F. Burk, 0. A. Levander, H. E. Ganther et al. (1-3). In particular, some authors (4-7) pointed out the importance of measuring the levels of this
element in whole blood and blood serum. In this paper, a method for the analysis of selenium in blood serum samples by proton-induced x-ray emission is described. The advantage of proton excitation has been widely discussed (8, 9). This technique permits realization of the required sensitivity (110ppb) without chemical preconcentration (IO).
EXPERIMENTAL Apparatus. The x-ray spectra were induced by 1.8-to 4-MeV proton beams -2 mm in diameter from the AN-2000 and CN Van de Graaff accelerators (High Voltage Engineering Corporation) installed at National Laboratories of Legnaro. Figure 1 shows the bombardment chamber and irradiation and detection geometry. A Si(Li) detector with an effective area of 30 mm2 and with steradians was used to detect a detection solid angle of 0.007.4~ the x-rays. The overall resolution of the standard electronic chain, ANALYTICAL CHEMISTRY, VOL. 49, NO. 9, AUGUST 1977
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