734
Anal. Chem. 1901, 53, 734-735
having almost identical separation characteristics.
ACKNOWLEDGMENT The active support and helpful suggestions of W. H. Griest are gratefully acknowledged. LITERATURE CITED Grob, K. Beitr. Tabakforsch. 1966, 3 , 403-408 Volimin, J. A.; Omura, I.; Seibl, J.; Grob, K.; Simon, W. Helv. Chim. Acta 1966, 4 9 , 1768-1778. Higgins, C. E.; Stokeiy, J. R. In "Tobacco Smoke Inhalation Bioassay Chemistry"; ORNL-5424; Oak Ridge National Laboratory: Oak Ridge, TN, Oct 1979. Griest, W. H; Higglns, C. E.; Caton, J. E.; Wike, J. S. "Abstracts of Papers", 180th National Meeting of the American Chemical Society, Las Vegas, NV, Aug 24-29, 1980; American Chemical Society, Washington, DC, 1980; ENVR B096. Bouche, J.; Verzeie, M. J. Gas Chromatogr. 1868, 6 , 501-505. Rutten, G. A. F.; Luyten. J. A. J. Chromatogr. 1972, 74, 177-193.
(7) Alexander, 0.; Garzo, G.; Paiyl, G. J. Chromatogr. 1974, 91, 25-37. (8) Grob, K. HRC CC, J . High Resolut. Chromatogr. Chromatogr. Commun. 1976, 1, 93-94. (9) Schomburg, G.; Dieimann, R.; Husmann, H.; Weeke, F. J . ChromatOgr. 1976, 122, 55-72. (10) Maskarinec, M. P., Oak Ridge National Laboratory, Oak RMge, TN, personal communication. (1 1 ) Ziatkis, A.; Lichtenstein, H. A,; Tishbee, A. Chromatographis 1973, 6 , 67-70.
RECEIVED for review July 21, 1980. Accepted December 22, 1980. This work Was supported by the Aerosol Research Branch, Atmospheric Chemistry and Physics Division, Environmental Science Research Laboratory, US. Environmental Protection Agency, R.K. Patterson, Project Officer, and the U.S. Department of Energy through IAG No. 40-691-78, EPA No. 78-D-X0228 under contract W-7405-eng-26 with the Union Carbide Corp.
Liquid Chromatographic Determination of Creatinine in Serum and Urine C. P. Patel" and R. C. George Analytical Laboratory, Research and Development Services, Ames Company, Divlsion Miles Laboratories, Xnc., P.O. Elkhart, Indiana 465 15
The assessment of creatinine levels in serum and urine is a valuable indicator in estimating renal function and establishing dose responses for new drugs. Until recently, creatinine in biological fluids has been assayed by using the well-known Jaffe alkaline picrate reaction. The specificity of the Jaffe reaction has been questioned in many publications, and so numerous modifications of this method have been published (1).
Recently, several investigators have shown the usefulness of liquid chromatography (LC) for the specific determination of creatinine free from other interfering serum components. These procedures have utilized cation exchange (2, 3) and reversed-phase (4-8) LC. This paper describes a LC procedure which utilizes isocratic conditions, ambient temperature, fixed-wavelength UV detection, normal-phase chromatography, and convenient sample preparation. This procedure has been proven to be useful for the routine analysis of clinical serum and urine samples for creatinine. A comparison study of this procedure with one of the widely used Jaffe methods ( I ) was performed.
EXPERIMENTAL SECTION Equipment. The chromatographic analysis was performed by using a liquid chromatograph, Model ALC/GPC 204, equipped with a Model 6000A solvent delivery system, a Model 440 absorbance detector, a WISP 710A autosampler, and a Model 730 data module integrator (Waters Associates, Inc., Milford, MA). A 5-pm silica column (4.6 mm X 25 cm), Zorbax SIL (DuPont Instruments, Wilmington, DE) was used with a guard column (No. 84550, Waters Associates). The precipitated serum protein was centrifuged in 1.5 mL. of polypropylene Eppendorf microcentrifuge test tubes (Brinkman Instruments, Inc., Westbury, NY) by use of a Fisher Model 59 centrifuge (Fisher Scientific Co., Pittsburgh, PA). Reagents. Methyl alcohol and acetonitrile were HPLC grade (Fisher Scientific Co.). Concentrated ammonium hydroxide, ACS reagent grade, 28%-30% NHBwas used. Clinical standard grade creatinine, Lot No. 914, was obtained from the U S . Department of Commerce, National Bureau of Standards, Washington, DOC. Quinine dihydrochloride monohydrate was obtained from Aldrich Chemical Co. (Milwaukee, WI). Samples, For method comparison, clinical serum samples obtained from the South Bend Medical Foundation (South Bend, IN) and commercially available lyophilized serum controls were 0003-2700/81/0353-0734$01.25/0
Box 70,
used. The source of urine samples was from apparently healthy adult individuals. For recovery studies, sterile human serum (Catalog No. 82-320-1, Research Products Division, Miles Laboratories, Inc., Elkhart, IN) was used. Procedure Alkaline Picrate Method. The procedure using Lloyd's reagent as described by Henry (1) was used without modification. Liquid Chromatography. The mobile phase was prepared by mixing 10.0 mL of concentrated ammonium hydroxide with 200 mL of methyl alcohol and 800 mL of acetonitrile. The mobile phase was degased under vacuum prior to use. A 100-bL sample (serum, controls, aqueous standards, or a 1:25 dilution of urine in distilled water) was transfered to an Eppendorf microcentrifuge test tube. Then 200 pL of acetonitrile containing 0.25 g/L of quinine dihydrochloride monohydrate (internal standard) was added and followed by mixing with a Vortex mixer. After 10 min, the sample was centrifuged at 7000 g for 5 min. The supernate was then transferred to an autosampler vial containing a limited volume insert (Part No. 72704, Waters Associates, Inc.) and 10 pL of the supernate was injected onto the column. Creatinine was eluted with the mobile phase at room temperature and a flow rate of 2.5 mL/min (1200 psi). The eluent was monitored at a wavelength of 254 nm with the detector sensitivity set at 0.05 AUFS and a chart speed of 0.25 cm/min.
RESULTS AND DISCUSSION Figure 1shows representative chromatograms of a serum creatinine sample of 9 mg/L, a urine sample diluted to a 55 mg/L creatinine level, and an aqueous 10 mg/L creatinine sample, respectively. The retention times for creatinine and the internal standard are 4.3 and 7.1 min, respectively. Several parameters were varied in obtaining this separation. These included the concentrations of acetonitrile, ammonium hydroxide, and methyl alcohol in the mobile phase, the flow rate, and the detector attentuation. The conditions chosen represent a good compromise among speed, resolution, and sensitivity. Linearity. The linearity of this method was studied by using (i) aqueous creatinine standards and (ii) samples prepared by the standard addition of aliquots of a creatinine solution (10 g/L creatinine in 0.01 N HC1) to pooled serum. The LC system response (ratio of area of the creatinine peak to the area of the internal standard peak) vs. concentration was linear up to the 160 mg/L creatinine level investigated. 0 1981 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 53,NO. 4, APRIL 1981 9 735 0.02
B
C
Table 11. Precision Study
B
0.01
day
E: $
1
&l
2
3 0
0.00
4
serum no. 1 2 3 4 5
amt of
creatinine av LC av % added, mg/L assay, mg/L recovery (9) 0 20.0 50.0 100.0
200.0
7.1 26.9 56.2 104.7 203.3
99.0 98.2 97.6 98.1
The results of the linear regression analyses were slope = 0.0048, y intercept = -0.0012, and r = 0.9998 for aqueous standards and slope = 0.0045, y intercept = 0.0432, and r = 0.9997 for spiked serum. Accuracy. Serum controls, e.g., Calibrate (General Diagnostics, Division of Warner-Lambert Co., Morris Plahs, NJ) and Moni Trol I and Moni Trol I1 (Dade, Division American Hospital Supply Corp., Miami, FL) were assayed to evaluate the accuracy of this method. The labeled values (43,51, and 14 mg/L, respectively) given for these controls by the manufacturer were based on the alkaline picrate method (as indicated on the product inserts). The alkaline picrate method is reported, however, to be nonspecific (8). Resdts of the LC analysis (38,47, and 10 mg/L, respectively), when compared with labeled values, therefore differed slightly as expected. the LC value may be considered more accurate due to the chromatographic procedure's specificity (3). Recovery. Aliquots of a pooled serum were spiked by adding various amounts of a creatinine solution (10 g/L creatinine in 0.01 N HC1). These samples were assayed in triplicate (Table I). Precision. Table I1 shows the result of a precision study, both day-to-day and within-a-day. Serums having low, normal, and high creatinine levels were assayed 10 times each day over a period of 5 days. The results given in Table I1 show good precision both within and between days. Comparison. This LC procedure was compared with Jaffe's alkaline picrate method (I) by analyzing 50 serum samples obtained from the South Bend Medical Foundation. The statistical data obtained from these analyses (slope = 0.93, y intercept -1.8, n = 50, r = 0.9938) revealed that the LC procedure gave results that were lower than those obtained
variation, %
10 10
7.6 11.7 17.0 7.5 11.5 17.4 7.6 11.6 17.1 7.5 11.5 17.3 7.6 11.6 17.6
0.37 0.39 0.61 0.37 0.54 0.48 0.28 0.52 0.56 0.34 0.49 0.59 0.26 0.57 0.80
4.9 3.3 3.6 4.9 4.7 2.8 3.7 4.5 3.3 4.5 4.3 3.4 3.4 4.9 4.5
10
10 3
Table I. Recovery Study
dev
10
CD
Flgure 1. Chromatogram of creatinine: (A) in serum, creatinine 9 mg/L; (B) in diluted urine, Creatinine 55 mg/L; (C) in distilled water, creatinine :O mg/L. CR = creatinine; IS = internal standard.
av, mg/L
10
5
10 10 10 10 10 10 10 10 10
std
coefficient
no. of analyses
of
by the alkaline picrate method. As mentioned earlier, this was to be expected if the LC procedure is considered to be more specific. Interferences. Substances that have been reported to interfere with Jaffe's alkaline picrate reaction were investigated. Many of these were insoluble either in the mobile phase or in a 50/50 (v/v) acetonitrile/distilled water mixture. Others were partially soluble but, when chromatographed, eluted along with the solvent front. The following substances also were found not to interfere at the respective concentration: 5 g/L of acetylsalicylic acid, ascorbic acid, tetracycline, and sodium heparin, 2.5 g/L of ampicillin and lithium acetoacetate; 1g/L of theophylline and caffeihe; 200 mg/L of phenylalanine, histidine hydrochloride, hydroxyproline, and tryptophan; 45 mg/L of bilirubuin; 100 mg/L of ethosuximide; 55 mg/L of phenobarbital; and 15 mg/L of primidone, phenytoin, and carbamazepine. We have not seen any interferences from the substances that, to our knowledge, might be found in normal clinical sera. Our work has shown that the silica packing (normal phase) did not show any sign of deterioration after performing analyses over a period of 12 months. The ammonium hydroxide in the mobile phase does not etch the silica as one might expect. LITERATURE CITED (1) Henry, R. J. "Clinical Chemistry: Principles and Technics,'' 1st ed.; Harper 8 Row: New York, 1964; pp 287-302. (2) Brown, N. D.; Slng, H. C.; Neely, W.; Koetitz, S. Clh. Chem. (Wlnston-Salem, N.C.) 1977, 23, 1281-1283. (3) Chiou, Win L.; Gadaila, M. A. F.; Peng, Geoffrey W. J . Pberm. Scl. w7a. ... -, 87. .. , ia2-ja7 .-- .- . . (4) Llm, C. K.; Richmbnd, W.; Robinson, D. 6.; Brown, S. S. J. Chrometogr. 1078, 143, 41-49. (5) Soldin, S. J.; HIII, J. G. Clh. Chem. (Wlnston-Salem, N.C.) 1078, 24, 747-750. (6) Buchanan, D. N.; TaR, K.; Domino, E. F. J . Chromtogr. 1070, 163, 212-216. (7) Krstulovlc, A. M.; Dziedzlc, L. B.; Caporusoo, J. M. Clln. Chlm. Acta 1979, 99, 189-194. (8) Splerto, F. W.; MacNeil, M. L.; Culbreth, P.; Duncan, 1.; Burtis, C. A. Clln. Chem. ( Winston-Salem. N.C.) 1080, 26, 286-290. (9) Nblsen, L. G.; Ash, K. 0. Am. J. Med. Techno/. 1078, 44, 30-37.
s.
RECEIVED for review November 14,1980. Accepted December 22, 1980.
