156
Anal. Chern. 1981, 53, 156-159
Strategies for Determination of Serum or Plasma Norepinephrine by Reverse-Phase Liquid Chromatography Gregory C. Davis‘ and Peter T. Kissinger” Department of Chemistry, Purdue Unlversiw, West Lafayetfe, Indiana 47907
Ronald E. Shoup Research Laboratories, Bioanalyticai Systems Inc., West Lafayetfe, Indiana 47906
Liquid chromatography wlth electrochemical detection (LCEC) Is shown to be capable of the determination of catecholamlnes In blood serum or plasma. Depending on the chromatogtaphlc condltions, two procedures for sample Preparation are applicable. Wlth moderate resolutlon columns, a two-step sample Preparation scheme based on lonexchange and alumina adsorptlon Is used prior to reversephase “lon-palr” LCEC with a thln-layer carbon paste electrode. Alternately, the hlgh resolution and unlque selectlvlty of a comrnerclally available reverse-phase materlal allowed a simpler extraction procedure, based on alumlna alone. Blood samples from sheep, rats, and humans were examined and found to be compatible wlth the LCEC methodology. I n contrast to radloenrymatlc assays, the procedures are rapid and Inexpensive. The use of Isotopes Is ellmlnated.
Unlike urinary catecholamine measurements, which represent an integration of sympathetic and central activity over a given period of time, serum catecholamine levels more accurately represent the temporal changes that can occur in the sympathetic nervous system. The measurement of circulating catecholamines, particularly norepinephrine, may well provide an important clinical monitor of certain diseases in which dysfunction of the sympathetic nervous system is implied (I). The lack of adequate analytical methods sufficiently sensitive for the quantitation of these compounds has hindered their diagnostic usefulness. Norepinephrine concentrations rarely exceed a few hundred picograms per milliliter of plasma or serum in healthy individuals. Epinephrine and dopamine concentrations are normally an order of magnitude lower than norepinephrine. Certainly the quantitation of these compounds is a challenging analytical problem. Fluorometric, radioenzymatic, gas chromatography, and gas chromatography/mass spectrometry (GC/MS) methods have all been employed to evaluate the serum or plasma levels (1). Two methods for liquid chromatography with electrochemical detection (LCEC) are described in this paper for determining serum catecholamines. These methods include the use of ion-pair reverse-phase chromatography to improve the selectivity of the separation. The choice of the proper sample preparation procedure depends largely on the selectivity of the stationary phase. The new methods have been applied to the determination of norepinephrine in human serum and plasma, rat serum, and sheep plasma. EXPERIMENTAL SECTION Apparatus. An LC-304 liquid chromatograph equipped with a TL-3 carbon paste electrode (BioanalyticalSystems Inc., West Present address: Monsanto Agricultural Products Co., St. Louis, MO 63166. 0003-2700/81/0353-0156$01.00/0
Lafayette, IN) was used throughout this work. The electrode potential was maintained at +0.750 V vs. a Ag/AgCl reference electrode,unless otherwise indicated in the discussion of figures. Several prepacked reverse-phase columns were evaluated for this work. These included 30 cm X 3.9 mm pBondapak CIB (Waters Associates, Milford, MA), 15 cm X 4.6 mm 5-rm Ultrasphere ODS (Altex Scientific Inc., Berkeley, CA), 25 cm X 4.0 mm 5-pm Spherisorb ODS (Phase Separations, Clewyd, United Kingdom), and 25 cm X 4.0 mm 5-rm Biophase ODS (Bioanalytical Systems). Mobile Phase. Acidic phosphate buffers with varying amounta of sodium octyl sulfate added for “ion pairing” were generally employed. For the Ultrasphere ODS column a pH 2.8 phosphate buffer (0.1 M)was used with 90 mg of sodium octyl sulfate added to each liter of buffer. The flow rate was 1.3 mL/min. The mobile phase used with the Spherisorb column was a pH 3.0 phosphate buffer (0.15 M) with 30 mg of sodium octyl sulfate added per liter. The flow rate was 1.5 mL/min. The separation on the Biophase column was optimized utilizing a mobile phase composed of 0.15 M monochloroacetate (pH 3.10), 1.0 mM disodium ethylenediarninetetraacetate, and 25 mg/L sodium octyl sulfate. The flow rate was 2.0 mL/min. Chemicals. GArterenol bitartrate (norepinephrine bitartrate), 3-hydroxytyramine hydrochloride (dopamine hydrochloride), bepinephrine bitartrate, and Uricase enzyme were obtained from Sigma Chemical Co. (St. Louis, MO). 3,4-Dihydroxybenzylamine hydrobromide was available from Aldrich Chemical Co. (Milwaukee, WI). Sodium octyl sulfate was supplied by Bioanalytical Systems. All other chemicals were reagent grade. Procedure I.The following isolation procedure is a modification of the method of Riggin and Kissinger (2) originally developed for urinary catecholamines and now optimized for serum norepinephrine. Two milliliters of serum, plasma, or appropriately spiked 0.1 M phosphate buffer (pH 7.0) was deproteinized with the addition of 200 pL of 4 M HC104,and an internal standard, 3,4-dihydroxybenzylamine,was added at this point if desired to improve precision (usually the case). The deproteinized samples were spun down on an Eppendorf centrifuge (Model 5412, Brinkmann Instruments, Westbury, NY) at 15000g for 5 min. A 1.6-mL aliquot of the supernatant was transferred to a 20-mL beaker. The pH was adjusted to 6.5 by the addition of 15 mL of pH 6.5 phosphate buffer (0.2 M). The samples were then poured onto cation-exchange columns (Bio Rex 70 (50-100 mesh), Bio Rad Laboratories, Richmond CA), and the effluent was discarded. The beakers were rinsed with 10 mL of water, and this was also added to the columns. Then 1.0 mL of 0.7 M H2S04 was applied to the columns. The effluent from the water and acid rinsings was not collected. Finally, 3.5 mL of 2.0 M NHdSO4 was applied to the columns. This eluate was collected in 5-mL conical glass screw-cap vials which contained 100 pL of a 10% solution of disodium ethylenediaminetetraacetate, 100 p L of a 4% metabisulfite solution, and 100 pL of uricase enzyme (optional). The vials were then briefly agitated. One milliliter of 3 M pH 8.6 Tris buffer was added to the vials followed by rapid addition of 100 mg of acid-washed alumina. The vials were capped and placed on a reciprocal shaker for 5 min. After the alumina was allowed to settle, the liquid was removed by aspiration, and the alumina was washed twice with water. The washings were removed by aspiration. Following the last wash, 0 1981 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 53, NO. 2, FEBRUARY 1981
the alumina was aspirated to near dryness. The catecholamines were eluted from the alumina by the addition of 500 pL of 0.2 M HC104. The vials were agitated on a vortex mixer and allowed to stand for 30 min. The acidic supernatant was filtered prior to injection using a Microfilter (Bioanalytical Systems) to remove fine alumina particles. One hundred microliters of the filtrate was injected onto the LC. In this procedure, dilution of the sample and synthetic standard by phosphate buffer help to minimize the differences between the two matrices. The absolute recovery for norepinephrine in the sample and the standard is therefore very nearly identical (60 f 5% relative standard deviation is typical for a given series of experiments when the internal standard is used). As a result, the relative recovery of the sample to the standard is -100% (>98 3 % relative standard deviation). The norepinephrine concentration in the sample is calculated by measuring the peak height of the sample and comparing that to the peak height of the phosphate standard taken through the procedure. In experiments of this type, quantitative absolute recovery (i.e., 100%), while desirable, is not necessary. High, reproducible relative recovery is the factor which establishes good precision. Procedure 11. Two-three milliliters of plasma or serum and 25 pL of 100 ng/mL dihydroxybenzylamine (in 0.1 M HC104) are combined in 5-mL conical glass screw-cap vials containing 100 pL of 10% ethylenediaminetetraacetic acid (EDTA) solution. Optionally, when assessing linearity, an additional 0-30 pL of a combined 75 ng/mL norepinephrine-35 ng/mL epinephrine solution (in 0.1 M HC10;) was added to replicate samples. Fifty milligrams of acid-washed alumina (AAO, Bioanalytical Systems, Inc.) was added to each vial. Then 1.0 mL of a pH 8.7, 1.5 M Tris buffer was added and the vial immediately capped, vortexed, and then shaken on a reciprocal shaker for 5 min. After the alumina was allowed to settle, the liquid was removed by aspiration and the alumina washed twice with water, using a polyethylene wash bottle and refilling the vial each time. After the last wash, approximately 0.5 mL of water was added and the alumina was transferred by use of a disposable pipet to a Microfilter (MF-l, Bioanalytical Systems) equipped with 0.2-pm RC58 membranes (Bioanalytical Systems). The samples were spun dry by centrifuging at 1600g for about 30 8. A clean, dry receiver tube was placed on the Microfilter and 200 pL of 0.1 M HC104was added to the sample compartment. After the mixture was vortexed briefly and centrifuged as before, the filtered extract was available for injection on the liquid chromatograph. For calibration purposes, 3.0 mL of 0.1 M phosphate buffer (pH 7.0) spiked with 25 pL of the 100 ng/mL dihydroxybenzylamine solution and 50 pL of the combined norepinephrine/epinephrine solution was assayed in duplicate along with every set of samples. The absolute recoveries for both this synthetic sample (67.4% for NE, 66.9% for E, 66.1% for DHBA) and spiked plasmas (65.9%, 65.5%, 70.4%) were virtually the same. Therefore, for calculation of sample concentrations, peak height ratios (relative to the internal standard dihydroxybenzylamine, DHBA) for plasma or serum samples were compared to peak height ratios for the synthetic btandard whose original concentrations were known. For norepinephrine (NE)
*
concn of NE in sample =
Sample Preparation. The sheep samples were obtained by drawing blood through a cannula located in the jugular vein of the animal. The blood was collected in heparinized collection tubes, and the plasma was separated from the cells by centrifugation. Human serum was obtained by venipuncture and was collected in serum separation tubes. The tubes were centrifuged, separating the serum from the cells. Freshly drawn human plasma was also obtained from the Central Indiana Ftegional Blood Center (Indianapolis) for examining matrix effects. Rat blood was collected from the animal following decapitation. The animal was rapidly inverted, allowing the blood to drain from the trunk into serum separation tubes through a glass funnel. As before, following a brief clotting time, the tubes were centrifuged to separate the serum. All of the samples were analyzed immediately in this
157
study. However, the samples are stable when frozen at -30 "C for several days with no sample treatment. If longer periods of time are expected between analysis and collection, then an antioxidant such as sodium metabisulfite should be added to the sample.
RESULTS AND DISCUSSION The determination of serum or plasma catecholamines by LCEC was not successfully accomplished until recently (3,4). This is primarily due to two factors. One is the continuing improvement in the efficiency of liquid chromatography columns. As the efficiency of the columns increases, the volume of mobile phase necessary to elute the compounds from the column bed decreases. The analyte is contained with a smaller volume, and the effective concentration of the compound as it passes through the detector is higher. The other factor is a better understanding of the electrochemical detector and the parameters that affect the signal-to-noise (S/N) ratio. Detection limits for the catecholamines in clinical samples have been extended to as low as 25 pg with a ES/N ratio of 5/1. The separations in two previous papers were performed on cation-exchange columns ( 4 4 ) . The results for norepinephrine were elevated somewhat compared to values obtained by other techniques, although it should be pointed out that the suhjed posture was unspecified. Our experience with plasma or serum has shown that even with the specific alumina isolation ]procedure, many oxidizable contaminants can be detected at the sensitivity setting necessary for low-level catecholamine determinations. Serum (or plasma) is a very complex sample matrix and is known to contain many electroactive components (e.g., cysteine, uric acid). In procedure I, the combination of the ion-exchange precolumns with alumina liquid/solid adsorption was implemented to remove as many extraneous compounds from the final solution as possible. The ion exchange lprecolumns isolate the cationic species (protonated catecholamines) from the sample. Following their elution, the catecholamines can be isolated further by making use of the well-established fact that at high pH, catechols form a stable complex with aluminum oxide. This allows catechols adsorbed on alumina particles to be washed of noncatechol compounds and potential electroactive contaminants. The catecholamines can be eluted from the alumina by the addition of dilute acid. Using the less selective cleanup procedure I1 required higher resolution reverse-phase columns to compensate for the greater complexity of the extract. A simple alumina extraction was sufficient, provided a 5-hm reverse-phase column of the proper selectivity was used. Uric acid, which has a concentration in normal human plasma or serum between 15 and 70 pg/mL (5),was the major contaminant species recovered when alumina was the sole isolation step. The high concentration and the polar nature of uric acid were responsible for its recovery through the alumina procedure. Even though the overall recovery of uric acid is low, in comparison to the catecholamines, it is usually the major peak in the chromatogram. The problem is compounded by the fact that the chromatographic characterif3tics of norepinephrine and uric acid are very similar (low k') for many commercially available reverse-phase columns. Ion- pair reagents can modify the stationary phase to increase the lz' of norepinephrine (6). Uric acid is generally unaffected by the presence of the anionic surfadant. For some columns (e.g, pBondapak CU), however, the separation was incomplete at any concentration of sodium octyl sulfate. The addition of uricase enzyme to the alumina isolation step can help to solve the uric acid problem. Uricase (80 m u ) was added to the sample as a reagent in the alumina adsorption procedure. The pH of the solution during the isolation is 8.6,
158
ANALYTICAL CHEMISTRY, VOL. 53, NO. 2, FEBRUARY 1981
I
I
UA
DHBA
NE
I
iOlnA f l i I
I
1
01
0
XI00
4
8
4
8
Flgure 1. Comparison of the alumina isolation procedure for noreplnephrlne in serum without (A) and with (6) addltion of the enzyme uricase to destroy uric acid. Waters pBondapak C-18 column (30 cm X 4.0 mm), applied potential +0.750 V vs. Ag/AgCI, CP-0 electrode material. DHBA
I
DHBA
li
j
, 6
1
16
0
0'
5
10
Figure 3. Norepinephrlne isolated from human plasma accordlng to procedure I. Biophase ODS 5-pm column (250 X 4.8 mm), applied potential 750 mV vs. Ag/AgCI, CP-W electrode material. The chromatogram on the left is for a reagent blank.
x2 x1 x2
0
rninutes
0
10
minutes
-x5
5
8
16
minutes
Figure 2. Plasma catecholamines obtained from 3 mL of plasma assayed according to procedure 11. Biophase ODs 5-pm column (250 X 4.6 mm), applled potential 4-650 mV vs. Ag/AgCI, CP-0 electrode
material. The reagent blank (phosphate buffer and internal standard)
Is shown on the right.
conveniently near the optimum for the enzyme. The enzyme is very effective in selectively oxidizing uric acid to allantoin, a nonelectroactive compound. Figure 1 demonstrates the difference between a sample that included uricase in the procedure and one that did not. It is apparent from the two chromatograms that the uric acid is significantly reduced. Unfortunately, however, the amount of norepinephrine in the two chromatograms is about 20-fold higher than would be observed in human plasma or serum. It is not unreasonable, however, for diseased individuals and for other species. While the use of uricase may be helpful in some circumstances, the authors favor avoiding this approach in favor of more elaborate sample preparation and/or higher resolution chromatography. Procedure I adapts both and procedure I1 incorporates only improved chromatography. The chromatograms of Figure 1 point out that there are still several unidentified oxidizable contaminants which are only eliminated when ion-exchange minicolumns are used in combination with the alumina. Another benefit of the minicolumn is that it removes uric acid prior to the alumina.
The advantages of a higher resolution stationary phase (procedure 11) are illustrated in Figure 2. A simple alumina extract of 3.0 mL of plasma was chromatographed on an efficient 5-pm spherical octyldecylsilyl-bonded silica material. The Biophase column was very flexible in this regard; the two components were easily resolved by varying the concentration of octyl sulfate in the 25-40 mg/L range. Absolute recoveries for each procedure were conducted with aqueous buffers as well as plasma samples. The latter were assayed both before and after a combined norepinephrine/ epinephrine spike (corresponding to 1 ng/mL) was added. The difference in peak heights between the unspiked and spiked plasmas was used to calcuate absolute recovery. Phosphoric acid has been shown to give a higher recovery of catecholamines desorbed from alumina (7) and was evaluated as a possible substitute for the HCIOl commonly used. The mean recovery (fstandard deviation) using procedure I was 67.1 f 4.0% (n = lo), but the void volume was considerably larger with this acid. This poses problems for measuring compounds with small k' such as norepinephrine. As a result, HCIOl was selected as the best eluent due to its low void volume response. The assay was applied to plasma and serum from several sources. Sheep plasma was obtained from the Purdue Animal Sciences Department. The blood was drawn from a single animal and enough plasma was acquired to be divided into eight 2-mL samples. Plasma norepinephrine was measured in the eight samples from an unstressed animal and the mean concentration was found to be 0.443 f 0.045 ng/mL (relative standard deviation 10.1%). The poor precision is accounted for by the fact that an internal standard was not used for the measurements. Norepinephrine and epinephrine were also isolated from a rat serum pool (six rats, blood collected by decapitation). The mean value of three determinations was 2.2 f 0.076 ng/mL (standard deviation) for norepinephrine and 5.0 f 0.14 ng/mL (standard deviation) for epinephrine. These values are in good agreement with Buhler et al. (norepinephrine 2.0 ng/mL and epinephrine 3.0 ng/mL) (8). The higher epinephrine values that were obtained in this study could be due to excessive restraint prior to decapitation. In the work by Buhler et al. they noted that epinephrine values can go as high as 24 ng/mL when the animals are restrained. Norepinephrine was also measured in human serum. The mean value of three determinations from a serum pool was 342 f 30 pg/mL. The pool was the combination of serum from four healthy males between the ages of 25 and 28. Figure 3 illustrates a chromatogram of norepinephrine isolated from human serum. Table I compares the human serum values obtained by this method with earlier reports. The values cited are for normal volunteers who were involved in clinical studies. The values are also classified by posture. The volunteers in this study were sitting during and prior to sample donation. The measured values for serum catecholamines are definitely
ANALYTICAL CHEMISTRY, VOL. 53, NO. 2, FEBRUARY 1981
Table I. Human Norepinephrine in Serum or Plasma in Normal Subjects amt norepinephrine,a Pg/d
subject posture
228 i: 24 (n -- 10) 510 i: 42 (n =: 10) 348 * 67 (n =: 18) 287 i: 20 ( n -- 44) 583 i: 28 (n = 44) 372 i: 42 (n = 12) 160 i: 20 (n =: 8) 270 i 40 (n =: 8) 174 i 52 (n =: 12) 240 i: 90 (n -- 50) 174 * 24 (n -. 12) 1166 “typical results” 668 * 27 (n =: 6) 342 i: 30 (n = 4) 252 “pool”
supine standing upright supine standing upright supine sitting supine supine supine unspecified unspecified sitting unspecified
methodb ref
R R R R R R
9 9 10 11
R R
F F F LCEC LCEC LCEC LCEC
11 12 13 13 14 15 16 17 3 c
4
a * standard deviation (n = number of samples). R= radioenzymatic; F = fluorometric; LCEC = liquid chromatography with electrochemical detection. Present work,
Table 11. Normalized Peak Current vs. Applied Potential for Rat Serum potential 9,NE @,EPI 750 mV standard 1.00 1.00 sample 1.00 1.00 650 mV standard 0.78 0.80 sample 0.80 0.83 550 mV standard 0.53 0.52 sample 0.49 0.54 450 mV standard 0.11 sample 0.12 a Applied potential at CP-0 working electrode vs. a Ag/AgCl reference electrode. Table 111. Normalized Peak Current vs. Applied Potential for Human Serum and Sheep Plasma potential”
900 mV standard human serum sheep plasma 830 mV standard human serum sheep plasma
Q.,NE
potentiala
790 mV standard human serum sheep plasma 750 mV 0.76 standard 0.78 human serum 0.82 sheep plasma a Applied potential at CP-W working electrode AgCl reference electrode. 1.00 1.00 ‘1.00
a, NE 0.60 0.59 0.68 0.12 0.15 0.14 vs. a Ag/
affected by postural changes (see Table I). One means of confirming the presence of compound when using LCEC is to generate a hydrodynamic voltammogram (HDV) for the chromatographic zone thought to contain that
159
compound. This curve is compared to an HDV for a stand,ard solution, and if the two curves match, then they very likely represent the same compound (18). One need not carry lout an entire potential scan but judiciously select three to four points over a suitable range. Table I1 presents the values for the rat serum study. Both the norepinephrine and epinephrine peaks were investigated. The relative peak current, 4, is the ratio of the peak heights to the peak height obtained at the most positive potential. Table I11 lists the values obtained for the human serum and sheep plasma for norepinephrine. The values are in good agreement, although the match is not as good as that for rat serum, due to the lower concentrations. As a result, more scatter in the data is introduced. One might note that in Table 111,the relative peak currents are slightly different than those of Table 11. This is due to the use of CP-W (wax binder) as an electrode material in the latter case as opposed to CP-0 (mineral oil binder11in the former. The two give slightly different electrode characteristics.
ACKNOWLEDGMENT We thank Richard Harner and Dennis Rasmussen for valuable technical assistance in sampling the subjects used for this investigation and James Keefe for this technical expertise in handling the several hundred samples examined in the exploratory stages of this study.
LITERATURE CITED (1) Davis, G. C.; Koch, D. D.; Kissinger, P. T.; Bruntlett, C. S.; Shoup, R. E. In “ Clinical Applications of Liquid Chromatography”; Kabra, P., Marton, L., Eds.; Humana Press: New York, In press. (2) Riggln, R. M.; Klssinger, P. T. Anal. Chem. 1977, 49, 2109-2’111. (3) Hallman, H.; Farnebo, L. 0.; Hemberger, B.; Jonsson, G. Llfe Scl. 1978, 23, 1049-1052. (4) Hjemdahl, P.; Daleskog, M.; Kahan, T. Llfe Scl. 1979, 25, 131-’138. (5) Faulkner, W. R.; Klng, J. W. In “Fundamentals of Clinical Chemistry"; Ttetz, N. W., Ed.; W. B. Saunders Co.: Philadelphia, PA, 1970; p 729. (6) Shoup, R. E. Ph.D Dlsseftatlon, Purdue University, West Lafayette, IN, 1980. (7) Felice, L. J.; Felice, J. D.; Klsslnger, P. T. J. Neurochem. 1978, 31, 1461-1465. (8) Bbhier, H. U.; DaPrada, M.; Haefely, W.; Plcottl, (3. 8. J . Phys~:/o/. (London)1978, 276, 311-315. (9) De Champlain, J.; Farley, L.; Couslneau, D.; van Amerlgen, M. R. Clrc. Res. 1978, 38, 109-114. (IO) Werdman, P.; Keusch. G.; Flammer, J.; Ziegler, W. H.; Renbi, F. C:. J . Clln. Endocrlnol. Metab. 1979, 48, 727-731. (11) Lake, C. R.; Ziegler, M. G.; Kopin, I. J. Llfe. Scl. 1976, 18, 1315- 1326. (12) Franco-Morselli, R.; Baudoinlegros. M.; Meyer, P. Clln. Scl. IMl. Med. 1978, 55, 9 7 ~ - 1 0 0 ~ . (13) Moerman, E. J.; Bogaert, M. G.; De Schaepdryver, A. F. Clln. Chlm. Acta 1976, 72, 89-96. (14) O’Hanlon, J. F., Jr.; Campuzano, J. C.; Howath, S. M. Anal. Biochem. 1970, 34, 568-581. (15) Grlffiths, J. C.; Leung, F. Y. T.; McDonald, T. J. Clln. Chlm. Acta 14870, 30, 395-405. (16) Renzini, V.; Brunori, C. A.; Vaiorl, C. Clln. Chlm. Act8 1970, 39, 587-594. (17) Allenmark, S.; Hedman, L. J. Llq. Chromatogr. 1979, 2 , 277-286. (18) Rice, J. R.; Klssinger, P. T. J . Anal. Toxlcol. 1979, 3. 64-66.
Received for review February 26,1980. Accepted Novemlber 3, 1980.
