Determination of creatinine in whole blood, plasma, and urine by high-performance liquid chromatography

ANALYTICAL

BIOCHEMISTRY

171, 135- 140 (1988)

Determination of Creatinine in Whole Blood, Plasma, and Urine by High-Performance Liquid Chromatography GANG-PING XUE,’ RICHARD C. FISHLOCK, AND ALAN M. SNOSWELL~ Department of Animal Sciences, Waite Agricultural Research Institute, University of Adelaide, Glen Osmond. South Australia 5064, Australia Received October 30, 1987 A sensitive method for the specific determination of creatinine in whole blood, plasma, and urine with high precision and accuracy is described. Samples were deproteinized by addition of acetonitrile and analyzed by high-performance liquid chromatography using a cation-exchange column with a mobile phase of 9% acetonitrile in 40 mM ammonium phosphate (pH 4.0). The recoveries of creatinine added to blood and plasma were almost complete, ranging from 99 to 101%. The coefficients of variation were very small, 1.6% for blood and plasma and 1.5% for urine. Samples can be assayed in 1 I-min intervals subsequent to the initial injection. As little as 2 ~1 of blood or plasma or 0.02 ~1 of urine is sufficient for chromatographic analysis. The present method was successfully used for the accurate measurement of small arterial-venous differences of creatinine concentrations in blood across body organs and showed that in the sheep creatinine is produced in the muscles and is excreted by the kidneys. The method is also suitable for routine analysis of Creatinine in clinical laboratories. 0 1988 Academic Press, IIIC. KEY WORDS: creatinine; blood; plasma; urine; high-performance liquid chromatography.

Measurement of creatinine levels in biological fluids is extensively used as an indicator of renal function. The method most widely employed for the determination of creatinine in clinical laboratories is a Jaffe alkaline-picrate procedure (1). However, many Jaffe-based methods can give overestimated values due to interference by endogenous and exogenous “pseudo-creatinine” chromogens formed in alkaline-picrate media, even when the procedure is adapted for continuous-flow assay with deproteinization or modified by Lloyd’s reagent or ether extraction (2).

Recently, several high-performance liquid chromatographic methods have been reported for the determination of creatinine in plasma, serum, and urine (3-8). These HPLC methods are more specific than Jaffebased methods. However, application of the known HPLC methods for the specific determination of creatinine in whole blood has not been reported. Thus, an HPLC method seemed appropriate for our study of creatinine metabolism in body organs, where the use of a sensitive and specific method for the determination of creatinine in blood with high precision and accuracy is critical for the detection of small arterial-venous differences of blood creatinine levels across body organs in studying the metabolism of creatinine in the whole body. This paper describes an HPLC assay for the measurement of creatinine in blood, as

’ Permanent address: Laboratory of Biochemistry, Zhejiang Agricultural University, Hangzhou, Zhejiang, The People’s Republic of China. * To whom correspondence should be addressed.

135

0003-2697188 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

136

XUE,

FISHLOCK,

well as plasma and urine, with very high precision and accuracy. Application of this method for the examination of the arterialvenous difference of creatinine concentrations in blood across organs is demonstrated. MATERIALS

AND

METHODS

Chemicals and solutions. Creatinine was obtained from Sigma Chemical Co. (St. Louis, MO.). Ammonia solution (33%, w/w) and phosphoric acid (93%, w/w) were of analytical-reagent grade, purchased from May and Baker (Melbourne, Victoria, Australia). Acetonitrile was of HPLC grade, obtained from Mallinckrodt (Clayton, Victoria, Australia). HPLC-grade water was prepared from double-distilled water by a reverse-osmosis water purification system (Sybron Bamstead, Boston, MA). All other chemicals were of the highest purity commercially available. Ammonium phosphate buffer (40 II’IM, pH 4.0) was prepared from ammonia solution and phosphoric acid. A 10 mM stock solution of creatinine was prepared by dissolving creatinine in 10 IIIM HCl and stored at -20°C. A series of creatinine working standards was made by dilution of the stock solution with water. Chromatographic system. The HPLC system consisted of a K25M solvent delivery pump, a K65B autosampler, a K95 variablewavelength uv detector (ETP Kortec Pty. Ltd., Sydney, N.S.W., Australia) and an SP4270 integrating chart recorder (SpectraPhysics Pty. Ltd., Bayswater, Victoria, Australia). A 25 X 0.46~cm i.d. cation-exchange Ultrasil CX (10 pm) column (Beckman Instruments (Aust.) Pty. Ltd., Gladesville, N.S.W., Australia) was used in combination with a 20 X 2-mm i.d. guard column packed with lo-pm Porasil (Waters Associates, Sydney, N.S.W., Australia). The columns were maintained at 28°C. The mobile phase consisted of 40 mM ammonium phosphate buffer (pH 4.0):acetonitrile (91:9, v/v) and was degassed under vacuum before use. The

AND

SNOSWELL

flow rate was 1.0 ml/min. The wavelength was 215 nm. Creatinine peaks were identified by their retention times and coelution with authentic standards and quantitated by comparing the peak heights of samples with those of authentic standards. The peak height measurements and baseline corrections were calculated and recorded using an SP4270 integrating recorder. The column was regenerated by washing with the following solutions in sequence: 20 ml of water, 60 ml of 80% methanol, 20 ml of water, and 60 ml of 0.5 M ammonium phosphate buffer (pH 2.4). Procedure. One hundred microliters of blood (blood cells were lysed by freezing and thawing), plasma, or 1:40 diluted urine in water was transferred to an Eppendorf microcentrifuge tube. Then 100 ~1 of water and 500 ~1 of acetonitrile were added and the samples were mixed with a Vortex mixer. After 5 min the samples were centrifuged in an Eppendorf microcentrifuge for 2 min. One hundred forty microliters of the supernatant was transferred to another Eppendorf microcentrifuge tube and evaporated to dryness in a Speed-Vat (Savant Instruments Inc., Hicksville, NY). It took less than 1 h for complete dryness. The dried samples were stable for at least several weeks, when stored at -20°C. Before analysis, the dried samples were dissolved in 100 ~1 ofwater and 20 ~1 of the solution (equivalent to 4 ~1 of blood or plasma or 0.1 ~1 of urine) was injected onto the column. For the calibration curve, 100 ~1 of a standard solution containing 2-80 nmol of creatinine was mixed with 100 ~1 of water or 100 ~1 of blood, plasma, or urine (I:40 dilution) and then 500 ~1 of acetonitrile was added. The assays were performed as described above. Alkaline-picrate method. The procedure using Lloyd’s reagent as described by Henry et al. was used for the colorometric determination of creatinine (9).

CHROMATOGRAPHIC

DETERMINATION

Animal Study, Three Merino ewes weighing about 60 kg were surgically prepared with multiple catheters and kept in metabolic crates as described previously (10). Five sets of blood samples were withdrawn from the aorta, pulmonary artery, renal vein, and inferior vena cava of each animal simultaneously at IO-min intervals and placed into heparinized tubes. The whole blood was stored at -20°C until taken for analysis. Blood samples were also taken from the jugular vein of normal adult Merino sheep (both sexes) and urine samples were collected from some of these sheep.

OF CREATININE

0;

031bli Retention

RESULTS AND DISCUSSION

Chromatography Creatinine is a weak base with a & value of 3.57 X 10-l’ at 40°C and is very soluble in water. Thus, by using these properties Chiou et al. successfully developed a cation-exchange HPLC method for the specific determination of creatinine in plasma, serum, and urine, using 0.1 M ammonium phosphate (pH 2.6) as the mobile phase (4). Application of their method in our laboratory for the determination of creatinine in whole blood samples resulted in overestimated values, compared to the values obtained by the alkaline-picrate method in combination with Lloyd’s reagent. This is probably because blood samples usually contain more interfering compounds than plasma, serum, or urine. Subsequently, we modified the mobile phase by first changing the pH and concentration of ammonium phosphate buffer and then introducing various concentrations (up to 10%) of acetonitrile or methanol into the ammonium phosphate buffer. The addition of acetonitrile or methanol to the ammonium phosphate buffer results in a separation on an ion-exchange column which is based upon the effects of both ionexchange and partition, thus altering the selectivity of the column. The retention time of the creatinine peak was reduced as the con-

137

time

0’ (mid

FIG. 1. Chromatograms of creatinine in (A) 4 fil of blood, (B) 4 pl of plasma, and (C) 0.05 ~1 of urine from sheep. Sample preparation and chromatographic conditions were as described under Materials and Methods. Arrows represent the positions of creatinine peaks.

centration of acetonitrile or methanol in the mobile phase was increased. Good resolution of the creatinine peak in blood samples was achieved with a mobile phase consisting of 9% acetonitrile in 40 mM ammonium phosphate buffer (pH 4.0). The addition of acetonitrile also improved the sharpness and symmetry of the creatinine peak and consequently the precision and sensitivity of the assay. However, substitution of acetonitrile with methanol in the mobile phase did not resolve the creatinine peak from other interfering compounds in blood samples. Figure 1 shows the chromatograms of blood, plasma, and urine samples, respectively. The retention time for creatinine was 8.3 min and it took 13 min for the last peak of a blood or plasma sample to be eluted. However, sample injection could be performed at 11-min intervals, since the last peak of a previous sample would not interfere in the quantification of creatinine in the subsequent sample. The column maintained its satisfactory resolution capacity for 250-400 injections. The complete regenera-

138

XUE, FISHLOCK,

tion of the column can be achieved by the washing procedure as described under Materials and Methods. Linearity

and Sensitivity

A linear relationship between the peak height and the creatinine concentration in blood, plasma, or urine was obtained for the ranges of concentrations tested (O-800 nmol/ml for blood and plasma and O-32 pmol/ml for urine). The correlation coefficients generally exceed 0.9999. We noticed that the calibration curve prepared by the addition of creatinine to water provides sufficiently accurate measurements as differences in the calibration curves prepared by the addition of creatinine to samples and to water were very small in this method. The lower limit for the detection of creatinine at a signal-to-noise ratio of 3 was about 3 nmol/ml for blood and plasma. As little as 2 ~1 of normal sheep blood or plasma or 0.02 ~1 of urine can be used for chromatographic analysis without loss of reproducibility or accuracy.

AND SNOSWELL

moving the organic solvent component from the deproteinized supernatant of a sample by drying and redissolving in water is desirable to achieve the high precision of the assay. Another advantage of this process is that volatile compounds are also removed from the samples, so that the possibility of interference caused by some volatile compounds is entirely eliminated. This procedure might be particularly valuable if a reverse-phase column is used for the determination of creatinine in biological samples. Recovery of creatinine was determined by analysis of blood and plasma samples spiked with standard creatinine at concentrations ranging from 40 to 400 nmol/ml sample. The recovery of added creatinine varied from 98.9 to 101.4%. Specificity

The purity of the creatinine peak was tested by comparison of the peak heights obtained at wavelengths of 215, 225, and 235 nm from aqueous creatinine standards with those of various samples. An identical uv abPrecision and Accuracy sorption pattern between the standard creatiThe precision of the method was assessed nine peak and the sample creatinine peak by repeated analysis of sample pools of sheep was observed at these three wavelengths. creatinine measured by the blood, plasma, and urine. The coefficients of Therefore, variation were 1.6% for both blood (n = 10) present method is likely to represent the true and plasma (n = 10) and 1.5% for urine (n creatinine concentration. The specificity of = 8). These values appear to be lower than this method was further tested by comparison with the most widely used alkaline-picthose of previously reported HPLC methods rate method in combination with Lloyd’s refor the determination of creatinine (3,4,6,7). One of the factors accounting for this high agent. Table 1 illustrates that the HPLC method gave lower values than those obprecision seems to be due to improvements method in the in our procedure of sample preparation. In tained by the alkaline-picrate of creatinine concentrations most other HPLC methods, after a sample is determination deproteinized with an organic solvent, the in blood, plasma, and urine. The relationship resulting supernatant is directly injected into between the two methods was further anaa column for analysis (3,4,6,7). Rapid evapo- lyzed for blood samples. The regression ration of the organic solvent-water mixture equation for the comparison between the reduring this operation would result in diffi- sults from the current HPLC method (y) culty in controlling an accurate volume of versus the alkaline-picrate method (x) was y sample for chromatographic analysis. Re- = 0.980x - 6.70 (n = 37, r = 0.956). Thus it

CHROMATOGRAPHIC

DETERMINATION TABLE

COMPARISON

1

BETWEEN THE HPLC METHOD AND THE ALKALINE-PICRATE METHOD CREATININE IN SHEEP BLOOD, PLASMA, AND URINE Blood

Sample No. 1

2 3 4 5 6

FOR DETERMINATION

Plasma

OF

Urine

HPLC

AP’

HPLC

AP”

HPLC

Apa

54.0 44.3 62.3 45.2 45.1 42.5

67.5 61.9 69.3 51.3 59.1 54. I

56.7 54.0 64.2 48.9

57.8 59.1 84.6 51.3

5112 6361 8015 7371

5579 6676 8790 7854

Note. Values are the average ’ Alkaline-picrate.

indicates specific.

139

OF CREATININE

of duplicate

that the present method

assays and expressed

is more

Application to the Analysis of ArterialVenousD@erence of Blood Creatinine Levels Study of arterial-venous differences of blood creatinine levels in animals has not been reported to date. Table 2 shows that the concentration differences of blood creatinine between aorta and various veins were very small. A sensitive and specific method with high precision is therefore highly desirable for

as nmol/ml.

the detection of real arterial-venous differences of creatinine levels. Using the present method, we successfully obtained new data about creatinine metabolism in body organs and tissues. It can be seen in Table 2 that creatinine levels in blood from the aorta of sheep were significantly lower than that in the inferior vena cava, but significantly higher than that in the renal vein. There was no significant difference in creatinine concentration between the aorta and the pulmonary artery. These results directly demonstrate that creatinine was produced in muscle and subsequently excreted by the kidneys.

TABLE 2 BLOOD

CREATININE

CONCENTRATIONS

IN VARIOUS

Creatinine Animal No.

1 2 3

Aorta

67.5 1 k 0.32 49.54 + 0.65 56.81 3~ 0.40

Pulmonary artery

67.96 f 0.63 49.82 + 0.59 56.56 + 0.34

concentration

BLOOD

VESSELS OF SHEEP

(nmol/ml)

Renal

vein

59.13 f 0.76“ 43.07 f 0.62” 48.41 f 0.36”

Inferior vena cava

70.44 f 0.72” 54.01 + 0.59” 61.24 + 0.50”

Note. Values are means + SE of five observations on each animal. Significance of differences was determined by comparison of creatinine concentration in the pulmonary artery, renal vein, and inferior vena cava with that of the aorta, using Student’s paired t test. “P < 0.01.

140

XUE, FISHLOCK,

In conclusion, we have developed a sensitive HPLC assay for the specific-determination of creatinine in blood, plasma, and urine with high precision and accuracy. The method is also simple and rapid and suitable for routine analysis in clinical laboratories. ACKNOWLEDGMENTS This study was supported by a grant from the AustraIian Research Grants Scheme (No. A086 16235).

REFERENCES 1. Boone, D. J. (1978) Proficiency Testing Summary Analysis for Clinical Chemistry (Survey II), Center for Disease Control, Atlanta, GA. 2. Narayanan, S., and Appleton, H. D. (1980) Clin. Chem. 26,1119-l 126.

AND SNOSWELL 3. Soldin, S. J., and Hill, G. J. (1978) Clin. Chem. 24, 747-750. 4. Chiou, W. L., Gadalla, M. A. F., and Peng, G. W. (1978)J. Pharm. Sci. 67, 182-187. 5. Spierto, F. W., MacNeil, M. L., Culbreth, P., Duncan, I., and Burtis, C. A. (1980) Clin. Chem. 26, 286-290. 6. Patel, C. P., and George, R. C. (198 1) And. Chem. 53,734-735. 7. Okuda, T., Gie, T., and Nishida, M. (1983) C/in. Gem. 29,85 l-853. 8. Brondeau, M. T., Simon, P., Bonnet, P., Guenier, J. P., and Ceauniz, J. De. (1987) J. Liq. Chromatogr. 10, 175-190. 9. Henry, R. J., Cannon, D. C., and Winkelman, J. W. ( 1974) Clinical Chemistry: Principles and Technits, 2nd ed., pp. 541-553, Harper and Row, Hagerstown, MD. 10. Robinson, B. S., Snoswell, A. M., Runciman, W. B., and Upton, R. N. (1984) Biochem. J. 217, 399-408.