Anal. Chem. 1985, 57,2365-2369 (13) Smith, D. A,; Heeg, M. J.; Heineman, W. R.; Eider, R. C. J . Am. Chem. SOC. lS84, 106, 3053-3054. (14) Hubbard, A. T.; Anson, F. C. I n “Electroanalytical Chemistry”, Bard, A. J., Ed.; Marcel Dekker: New York, 1970; Voi. 4, pp 129-214. Teo, B.-K. J . Am. Chem. SOC. 1981, 103, 3990-4001. Mullica. D. F.: Milliaan, W. 0.; Garnier, R. L. Acta Crystallogr.,Sect. 8 1980, 8 3 6 , 256112564. Figgis, B. N.; Skeiton, B. W.; White, A. H. Aust. J . Chem. 1978, 3 1 , 11F15-llF19 . . - . .- - . Fletcher, S. R.; Gibb, T. C. J . Chem. Soc., Dalton Trans. 1977, 309-316. Beall, G. W.; Milligan, W. 0.; Korp, J.; Bernal, I.; McMuiian, R. K. Inorg. Chem. 1977, 16, 207-209. Swanson, 8. I.; Ryan, R. R. I n w g . Chem. 1973, 12, 283-286. Herren, F.; Ludi, A.: Fischer, P. Acta Crystallogr.,Sect. 8 1979, 835, 3 129-31 30. Figgis, B. N.; Gerloch, M.; Mason, R. Proc. R . SOC. London, Ser. A 1969, 3 0 9 , 91-118. Bailey, W. R.; Williams, R. J.; Milligan, W. 0.Acta CrYStallogr., Sect. B 1973, 8 2 9 , 1365-1368. Tullberg, A,; Vannerberg, N.-G. Acta Chem. Scand. 1974, A28, 551-562. Gravereau, P.; Gamier, E.; Hardy, A . Acta Crystallw., Sect. 13 197% 835,2843-2848. Morosin, B. Acta Crystallogr ., Sect. 8 1978, 834, 3730-3731.
-
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(27) Taylor, J. C.; Mueiier, M. H.; Hitterman, R. L. Acta Crystallogr. , Sect. A 1970, A26, 559-5167, (28) Beall, G. W.; Mullica, D. F.; Mllligan, W. 0.; Korp, J.; Bernai, I. Acta Crystallogr., Sect. B 1978, 834, 1446-1449. Oliver, J. D. Inorg. Nucl Chem. Lett. (29) Mullica, D. F.; Milligan, W. 0.; 1979, 15, 1-5. (30) Kiriyama. R.; Kiriyama, H.; Wada, T.; Niizeki, N.; Hirabayashi, H. d . Phys . Soc. Jpn . 1964, 19, 540-549. (31) . , Shaw, C. F., 111; Schaeffer, N. A.; Elder, R. C.; Eidsness, M. K.; Trooster, J. M.; Cab, G. H. M. J . Am. Chem. SOC. 1984, 106, 35 1 1-3521. (32) Vanderheyden, J.-L.; Ketring, A. R.; Libson, K.; Heeg, M. J.; Roecher, L.; Motz, P.;Whittle, R.; Elder, R. C.; Deutsch, E. Inorg. Chem. 1984, 2 3 , 3184-3191.
RECEIVED for review February 7,1985. Accepted May 23,1985. Support for this project was provided by NSF Grants PCM8023743 (R.C.E.) and CHE8401525 (R*CaEand W*RaH*) and a University Research Council Grant from the University of Cincinnati. The authors acknowledge the use of SSRL, which is operated by the Department of Energy.
Isolation of Plasma Components by Double Antibody Precipitation and Filtration: Application to the Chromatographic Determination of Arbaprostil [ (15R)- 15-Methylprostaglandin E2] J. W.Cox,* R. H. Pullen, and M. E.Royer Drug Metabolism Research, T h e Upjohn Company, Kalamazoo, Michigan 49001
The utlllty of a double antibody preclpltatlon method for the selectlve extractlon of plasma components was tested for abraprostil, an antiulcer prostaglandin. Plasma samples ( 1.O mL) were treated successlvely wlth rabblt anti-arbaprostli serum and goat anti-rabblt serum. The Immunoprecipitate was collected by flltratlon, washed with water, and extracted wlth ethyl acetate to recover the drug In 60% overall yield. After internal standard was added, the extract was derivatlzed wlth panacyl bromide and analyzed by column switchlng hlgh-performance llquid chromatography (HPLC) wlth fluorescence detection. Standard curves of HPLC peak helght ratlo vs. plasma concentration were linear to 200 pg/mL and the assay quantltatlon limit was 25 pg/mL (signal-to-noise ratio 6:l). The lnterassay reiatlve standard deviation at 119 and 60 pg/mL was 6 and 13%, respectively. The method was used to quantify arbaprostil in dog plasma following intravenous administration. Compared to an alternatlve arbaprostll plasma cleanup procedure uslng solid phase extractlon and chromatographic puriflcation, the immunoprecipitation procedure was faster, simpler, and provided an equally acceptable chromatographlc base line.
Interferences in liquid/liquid or solid phase extracts of biological samples often prevent the direct chromatographic determination of trace substances, and intermediate chromatographic cleanup can be required. This is especially true of prostaglandins (PG’s) because they are typically present in nanogram per milliliter concentrations or less and are physicochemically similar to naturally occurring compounds present in higher concentration. Extraction methods for P G s based on liquid/liquid ( I , 2) or solid phase (2-4) partitioning
are relatively nonselective and the extracts are frequently unsuitable for direct analysis, even by highly specific gas chromatography/mass spectrometric (GC/MS) quantitation methods (5-9). The necessity of purifying extracts before chromatographic analysis makes the analytical procedures more complex, laborious, and time-consuming to develop. Facing a similar problem for the determination of steroids in physiological fluids, Gaskell et al. took advantage of immunoadsorption techniques (10-12) to simplify extraction and cleanup procedures for GC/MS analysis (13-16). Plasma or serum samples were treated with solid phase coupled antisera and the antibody-bound steroids were separated from unbound components by centrifugation and washing. The steroids were then extracted from the antibodies with methanol. The authors reported that the selectivity of the immunoadsorption procedures permitted the direct analysis of extracts and eliminated the need for intermediate chromatographic cleanup steps. Royer and KO recently described an alternative immunoextraction method in which alprazolam, a triazolobenzodiazepine, was isolated from serum by double antibody precipitation and centrifugation (17). Both of these strategies exploit the inherent selectivity of antibody-antigen interactions to obtain relatively pure sample extracts. The utility of the immunoextraction concept for PG analysis was tested with a double antibody precipitation and filtration method for the extraction of arbaprostil, a gastric antisecretory and cytoprotective agent (18,19). A high-performance liquid chromatography (HPLC) method for arbaprostil quantitation was recently described in which solid phase plasma extracts were fractionated by reversed-phase HPLC before analysis (20). The fraction containing arbaprostil was then derivatized and analyzed with a normal-phase column switching HPLC system (20,21). As demonstrated by the present work, the sample preparation procedure for arbaprostil analysis can be
0003-2700/85/0357-2365$01.50/0 0 1985 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 12, OCTOBER 1985
substantially simplified through the use of the immunoextraction technique without detrimentally affecting the performance specifications of the analytical method. EXPERIMENTAL SECTION Materials. Arbaprostil [(15R)-15-methyl-PGEz],(15R)-1515R)-15-methyl-PGEz(specific methyl-5,6-truns-PGEz,[ll@-3H]-( activity 3.5 mCi/mg), and panacyl bromide [p-(9-anthroyloxy)phenacyl bromide] were supplied by Pharmaceutical Research and Development, The Upjohn Co. (Kalamazoo, MI). Rabbit anti-arbaprostil serum was supplied by F. A. Fitzpatrick and M. A. Wynalda (Lipids Research, The Upjohn Co.). Goat anti-rabbit serum was provided by D. P. Kane (Upjohn Diagnostics). Extraction solvents were UV or HPLC grade from Burdick and Jackson Laboratories (Muskegon, MI). Plasma Standard Preparation. Blank human plasma was obtained from Plasma Alliance (Knoxville, TN). Blood was collected from individual donors in 1.2-L lots using plastic containers and sodium citrate anticoagulant. The plasma was separated and stored at -20 "C. Blank dog plasma was collected by jugular venipuncture into all-plastic Monovette 10-mL EDTA tubes (Sarstedt, Inc., Princeton, NJ). The tubes were placed on ice and centrifuged within 30 min of collection at 15OOg for 30 rnin at 4 "C. The plasma was separated and stored at 20 "C in 20-mL glass scintillation vials. An accurately weighed amount of arbaprostil was dissolved in acetonitrile to produce a stock concentration of 75.0 pg/mL. This solution was diluted serially to produce standard working solutions using all polypropylene materials. Aliquots (3.0 mL) of plasma in 10-mL polypropylene tubes were then spiked with the standard solutions, keeping the acetonitrile concentration of the resulting plasma standards less than 1% (v/v). Five standards were prepared over a concentration range of 25-200 pg/mL. After mixing, the plasma standards were capped and stored at -20 "C. Plasma standards containing radioactive arbaprostil were prepared by the same spiking method. Prostaglandin Immunoprecipitation. Frozen plasma samples were thawed and filtered through a 0.45-pm Millex HV filter unit (Millipore Corp., Bedford, MA). Rabbit anti-arbaprostil serum (0.30 mL of a 1to 50 dilution in 0.1 M Tris-HC1 buffer, pH 7.8, containing 0.1 mg/mL of thimerosal) was added to 1.0 mL of filtered plasma in a 12 X 75 mm polypropylene tube and the tubes were shaken on a horizontal shaker (Eberbach Corp., Ann Arbor, MI) at 3 cycles/s for 30 min. Goat anti-rabbit serum (0.10 mL of a 1 to 5 dilution in 0.1 M Tris-HC1 buffer as above) was then added and the tubes were vortexed for 15 s and incubated overnight at 4 "C. Immunoprecipitate Isolation and Extraction. After overnight incubation, samples were vortexed for 30 s and then filtered through 70-pm polyethylene filters to remove large (nonimmune) particulate matter. Filters were prepared by inserting a filter frit (3/8 in. diameter; Analytichem International, Harbor City, CA) into a 4-mL syringe barrel reservoir (Analytichem International). The filters were placed on a vacuum manifold (J.T. Baker, Phillipsburg, NJ) and the samples were filtered by suction (660 mmHg internal manifold pressure) into 12 X 75 mm polypropylene tubes followed by a 0.25-mL water wash. The immunoprecipitate was isolated by filtering the 70-pm filtrates through a Whatman GF/F microfiber filter mat (Whatman Laboratory Products, Inc., Clifton, NJ) mounted on a cell harvester equipped with a suction head for 12 12 X 75 mm tubes (Skatron, Inc., Sterling, VA). The filter mat (4 X 10 in.) was prepared for the filtration by immersing in ethyl acetate for 1h and then allowing to air-dry. After the filter was wetted with water, 12 samples were simultaneously vacuum filtered (5-10 mmHg) through individual filter disks. The filters were then washed with seven 3-mL portions of distilled water and cut from the mat using a filter disk transfer system (Skatron) into 14 X 58 mm polyethylene scintillation vials (Skatron). Ethyl acetate (1.0 mL) was added to each vial and the vials were shaken at 3 cycle/s on a horizontal shaker for 15 min to extract arbaprostil. The extracts were then transferred with a 0.5-mL wash of ethyl acetate to 12 X 75 mm polypropylene tubes using a polypropylene tipped pipettor. Quantitation of Arbaprostil in Immunoextracts by HPLC. Internal standard [(15R)-15-methyl-5,6-truns-PGE2, 384 pg, 12
pL of a 32 ng/mL solution in acetonitrile] was added to each extract and the solvent was evaporated at 40 "C under a stream of nitrogen. The residue was reconstituted, derivatized with panacyl bromide, and analyzed by heteromodal column switching HPLC with fluorescence detection (19). Data from the analysis of fortified plasma standards were analyzed by unweighted linear regression best-fit of peak height ratio (arbaprostil/IS) vs. concentration. Dog Pharmacokinetic Study. A 13-kgpurebred beagle was fed a normal chow meal at 6 am and 2 h later given a 39-pg dose of (15R)-15-methyl-PGEz(30 pg/mL solution in ethanol/sterile saline, 5/95, v/v) via the right cephalic vein. Blood samples were collected from the left cephalic vein with a Butterfly infusion set (Abbott Hospitals, Inc., North Chicago, IL) at predose, 5,10,15, and 30 min and by jugular venipuncture at 1 and 2 h into Monovette 10-mL EDTA tubes. The tubes were placed on ice and centrifuged to separate plasma within 30 min of collection. The plasma was withdrawn with a polypropylene tipped pipettor and stored at -20 "C in 10-mL polypropylene tubes with push-caps. RESULTS Optimal Antibody Dilution for Arbaprostil Immunoprecipitation. The conditions for immunoprecipitation of arbaprostil were established for a 700 pg/mL concentration of drug, which is roughly 3-fold higher than the expected maximal concentration in human plasma following a 50-pg oral dose (20,22). The concentrations of the primary and secondary antisera were independently varied in a series of experiments using radiolabeled drug. The immunoprecipitate was separated by centrifugation (10 rnin at 1500g) and the precipitation efficiency was determined by counting both the suspended pellet and supernatant. Maximal efficiency was achieved with a 1to 233 dilution of the rabbit anti-arbaprostil serum and a 1 to 70 dilution of goat anti-rabbit serum. The precipitation efficiency of plasma radioactivity was 90 f 2% (standard deviation, n = 6) and was constant over a 100-700 pg/mL concentration range. Approximately 9% of the drug in uncentrifuged samples adhered to the polypropylene tubes when decanting and was not recoverable by vortexing with water. The practical immunoprecipitation efficiency for subsequent processing steps was therefore 81% . Immunoprecipitate Filtration. Whatman GF/F glass microfiber filters efficiently retain double antibody precipitates (23). Glass microfiber filters with retention ratings of 1.0 pm and greater gave poorer recovery of immunoprecipitates and also exhibited greater variability in recovery (23). Similarly, we found that several membrane filters (cellulose acetate, cellulose nitrate, and polytetrafluoroethylene) with 0.45-pm ratings retained the radiolabeled arbaprostil immunoprecipitate but that the recovery was poorer with 0.8- and 1.0-pm filters. For filters which retained the immunoprecipitate, the filtration rate decreased as the amount of precipitate increased. Glass microfiber GF/F filters had the highest capacity to retain the precipitate without clogging and, consequently, were selected for further work. Preliminary experiments indicated that the overnight incubation of plasma samples resulted in the formation of a small fibrinaceous clot which blocked the GF/F filters and prevented filtration. For the present study, the clot was removed by routinely filtering samples through a 70-pm polyethylene frit. Approximately 3% of the antibody bound radiolabeled PG was lost in this step. Subsequent experiments have shown that clot formation could be effectively prevented by adding 2 mg of NazEDTA to each sample and by shortening the incubation period to 3 h. Neither of these changes affected the precipitation efficiency of the PG. After 70-pm filtration, the samples were filtered through GF/F filters using an instrument designed to simultaneously filter 12 samples through individual filter disks. For the initial evaluation of filtration recovery of the immunoprecipitate, plasma/antiserum mixtures containing radiolabeled arba-
ANALYTICAL CHEMISTRY, VOL. 57, NO. 12, OCTOBER 1985 a -
Table I. Extraction Recoveries of Radiolabeled Arbaprostil from Immunoprecipitates Retained on Glass Microfiber Filter Disks Using Various Organic Solvents (See Text for Extraction Conditions)
extraction solvent acetonitrile tetrahydro-
recovery? %
solvent
extraction recovery: %
100 99
butyl chloride toluene chloroform methylene chloride ethylene dichloride
29 28 24 22 19
furan
2-Propanol ethyl acetate methanol acetone
99 96 95 63
b -
-.
400
aRecoverie~were calculated as a percentage of the amount of antibody bound prostaglandin available at the time of GF/F filtration, which was approximately 78% of the prostaglandin in a 1.0-mL Dlasma samde. prostil were filtered and the disks were washed with 6.0 mL of water and cut and desposited into scintillation vials. One milliliter of water and 15 mL of liquid scintillation cocktail were added and the vials were shaken on a horizontal shaker for 1h. The recovery of plasma radioactivity from the filters was 77 f 4% (standard deviation, n = 4) over a 180-730 pg/mL concentration range. This corresponded to 99% of the antibody bound PG that was available for filtration. Less than 0.5% of radiolabeled arbaprostil was recovered from the filters in the absence of the primary antiserum, indicating that adsorptive binding by the filter was insignificant. The influence of wash solvent volume on recovery was investigated by washing identical 500 pg/mL samples in groups of six with 6,9, and 21 mL of deionized water. There was no significant difference in recovery 0, > 0.05) between the groups. Thus, wash solvent volumes of a t least 21 mL could be used to remove nonspecifically retained plasma components from the filter without significantly affecting the recovery of arbaprostil. Extraction of Arbaprostil from the Immunoprecipitate. Several solvents were tested for extraction efficiency of arbaprostil from the immunoprecipitates retained on GF/F filter disks. Antisera were added to identical plasma samples containing 500 pg/mL of radiolabeled arbaprostil and the immunoprecipitates were collected on filter disks, washed with 6 mL of water, and then cut and deposited into polyethylene vials. One milliliter of solvent was added (sufficient to cover the disk) and the vials were shaken on a horizontal shaker for 30 min, after which the solvent was transferred to scintillation vials, evaporated, and counted. Maximum recoveries of the available antibody bound radioactivity (95-100%) were obtained with 2-propanol, methanol, tetrahydrofuran, acetonitrile, and ethyl acetate (Table I). Several of these solvents caused significant filter degradation that was noticeable as a white residue remaining after evaporation of the extracts. Ethyl acetate caused the least amount of filter degradation and consequently was selected as the solvent for routine extractions. In a time course recovery experiment, it was found that the recovery of radioactivity in ethyl acetate extracts of immunoprecipitates on filter disks reached a maximum value after 5-10 min of shaking and remained constant to at least 45 min. High-Performance Liquid Chromatographic Analysis of the Immunoprecipitate Extract. Internal standard [(15R)-15-methyl-5,6-trans-PGE,1 was added to the immunoprecipitate extract and it was derivatized with panacyl bromide to form fluorescent panacyl esters of the prostaglandins, which were then determined by analysis on a normal phase, column switching HPLC system (20,211. No peaks with retention times in the region of arbaprostil were detected in blank human plasma immunoprecipitate extracts (Figure
2367
0
\
--1
t
t
.
Time (min) Flgure 1. HPLC chromatograms of blank human plasma processed by: (a) C18/Si two-stage extraction (79);(b) double antibody precipitation with isolation and washing (5 times) by centrifugation; and (c) double antibody precipitation with isolation and washing by filtration.
Arrows indicate the elution time of arbaprostil. Plasma 180 pg/mL Standard
Plasma 25 pg/mL Standard
Plasma Blank
4001 Arbaprostil
I )I
35
39 Time (min)
35
39 Time (mini
35
39
Time
(min)
Flgure 2. Representative chromatograms from the HPLC analysis of fortified human plasma double antibody filtration immunoextracts. IS = internal standard.
IC);however, dog plasma extracts contained a component with the retention time of arbaprostil a t an equivalent concentration of approximately 10 pg/mL. For purposes of comparison, Figure 1 also includes chromatograms of human plasma extracts prepared by sequential C18/Si BondElut extraction (20) (Figure l a ) and by double antibody immunoextraction using centrifugation rather than filtration (Figure Ib). In the latter case, the immunoprecipitate was washed five times by repeated centrifugation and resuspension in 1.0 mL of water and finally extracted by shaking with methanol to recover the PG. Most of the interfering components in plasma were removed by the immunoextraction-centrifugation method after only two washes but could not be consistently eliminated with additional washes. The filtration method provided a relatively convenient means to reproducibly wash the immunoprecipitate and it consistently produced chromatographically acceptable extracts from several plasma donors. Extraction recovery from the filter disks was demonstrated to be independent of concentration over a range of 25-180 pg/mL in human plasma by HPLC analysis of four sets of fortified plasma standards. The best-fit linear correlation coefficient ( r ) for standard curves of peak height ratio (arbaprostil/IS) vs. plasma concentration over this range was 0.99 or greater and the y intercepts were not significantly different from zero ( p > 0.05). Fortified plasma standard chromato-
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Table 11. Precision and Accuracy of the Arbaprostil Plasma Immunoextraction HPLC Assay Proceduren fortified plasma source
concn,
pg/mL
mean assay result, pg/mL
I
1000 \
% re1 std
dev (n)
% error
human 119 127 6.2 (9) 6.7 human 59.6 63.3 12.5 (7) 6.2 99.8 98.7 13.2 (7) -1.1 dog 24.7 24.6 18.0 (7) dog -0.4 OPlasma samples were analyzed in duplicate or triplicate on three different days to determine both intra- and interday method variance. Analysis of variance procedures indicated that there was no significant interdgy difference in assay results (p 2 0.05) and so the data were Dooled.
m l Q
v
C
=E0
100
I
C
Q 0
s
10
0
10
20
30
40
50
60
~
grams are presented in Figure 2. The interassay mean relative
weight response (RWR) factor for arbaprostil (peak area ratio/arbaprostil concentration/internal standard concentration) was 0.56 f 0.04 (standard deviation, n = 20). Since the observed RWR factor in solution standards was not significantly different from 1.0, the plasma standard RWR indicated that the extraction recovery of arbaprostil from human plasma was 56 f 4%. Similar results were obtained from the analysis of three sets of fortified dog plasma standards, for which linearity was demonstrated from 25 to 200 pg/mL (r 2 0.994) and the mean RWR was 0.63 f 0.03. The precision of the analytical procedure was evaluated at 60 and 119 pg/mL levels in human plasma and at 25 and 100 pg/mL levels in dog plasma by repeating replicate assays of fortified plasma on 3 days. The results of these experiments are presented in Table 11. There was no significant interday difference in assay results a t these concentrations. The assay quantitation limit when processing 1 mL of plasma was 25 pg/mL (signal-to-noise ratio 6:1), at which the assay relative standard deviation in dog plasma was 18%. The utility of the analysis procedure was demonstrated by determining the concentration of arbaprostil in dog plasma following a 3 pg/kg iv, bolus dose. High concentration samples were diluted with blank dog plasma so that their concentrations were 50-150 pg/mL at the time of immunoprecipitation and were assayed in duplicate. A semilogarithmic plot of arbaprostil plasma concentration vs. time is presented in Figure 3.
DISCUSSION The primary antiserum used for this double antibody immunoextraction was highly specific for arbaprostil, showing minimal cross reactivity with its (15S)-epimer (0.2%), (15R)-15-methyl-PGA2(1.3%), (15R)-15-methyl-PGB2(0.6%), and 20.02% with the major metabolites of arbaprostil and other structurally relevant ligands (24). It should be noted, however, that a high degree of antibody binding specificity is not a prerequisite for using the antibody for immunoextraction purposes, so long as the cross-reacting ligands do not interfere with quantitation. Whereas moderate levels of cross reactivity can cause considerable inaccuracy in immunoassay results unless the sample is appropriately fractionated prior to analysis, immunoextraction recovery can be desensitized to the effects of competitive binding by adding excess antibody. In the present case, the extraction conditions were established so that the extraction recovery was constant to a t least 700 pg/mL, even though the assay linearity did not need to exceed 200 pg/mL. Furthermore, in certain cases, cross reactivity of antisera with selected structurally related materials could be a desirable feature, as when the analytical method is capable of quantifying both a drug and its metabolites. In the present case, for example, if the antiserum had shown cross reactivity with ( 15S)-15-methyl-PGEz, it
Time (min) Figure 3. Semilogarithmic plot of dog plasma concentrations of arbaprostil vs. time following a 3 pg/kg lv bolus dose as determined by HPLC analysis of plasma immunoextracts.
would have been both possible and desirable to coextract both epimers for chromatographic determination (20, 21). The antibody-arbaprostil complex was separated from solution by double antibody precipitation. Centrifugation and filtration methods were both tested as a means of isolating the precipitate, but preliminary experiments indicated that the centrifugation route failed to produce chromatographically acceptable extracts with a reasonable number of washes (Figure lb). This may have been because it was difficult to obtain a fine suspension of the precipitate for efficient washing, with the result that there was irreproducible carryover of interfering substances. This problem may be unique to arbaprostil, however, because of the extremely high concentration of substances interfering with arbaprostil measurement. Subsequent work has demonstrated that double antibody centrifugation procedures are capable of providing chromatographically acceptable extracts for PGEl and (158)-15-methyl-PGE2. The filtration method was effective at separating arbaprostil from chromatographically interfering substances (Figure IC). The filtration recovery of the immunoprecipitate from prefiltered plasma was quantitative, and the stability of the antibody complex in deionized water permitted thorough washing of the retained precipitate without significant loss of the PG. The radiolabeled drug was then recovered in nearly quantitative yield from the filter disks by shaking with ethyl acetate. On the basis of radioactivity results, the overall recovery of arbaprostil from plasma with this procedure was 77%, in contrast to the 60% recovery indicated by HPLC analysis of the extracts. Possible explanations for this difference are that some of the extracted radioactivity remained bound to solubilized or suspended antibody and was unavailable for derivatization or that some degradation occurred during the immunoextraction procedure. Despite the discrepency, the linearity of the HPLC peak height ratio with standard concentration was good over the concentration range of interest (r 2 0.99, upper limit 180-200 pg/mL). In a previously described procedure for the quantitation of arbaprostil in plasma, solid phase extracts of plasma were purified by reversed-phase HPLC before eventually quantifying arbaprostil by column switching HPLC (20). Compared with this method, the immunoextraction procedure was both faster and simpler. The time to process 36 samples by double antibody precipitation-filtration was approximately 4 manhours in addition to an overnight incubation period, in contrast to the 6 man-hours required to process 20 samples by the HPLC cleanup procedure. The performance specifications of arbaprostil plasma assay methods using either immu-
ANALYTICAL CHEMISTRY, VOL. 57, NO. 12, OCTOBER 1985 ~~
Table 111. Comparison of Performance Specifications for Arbaprostil Human Plasma HPLC Assay Procedures Using Immunoextraction or Solid Phase Extraction-HPLC Cleanup Techniques assay specification
immu-
solid phase extraction-
performance parameter
noextraction”
cleanupb
extraction-cleanup recovery (%) linearity (pg/mL) precision (70RSD a t 50-60 p g / m L ) sensitivity (pg/mL, SIN = 6)
56 25-180 12.5 26
a W h e n processing 1 mL o f plasma. plasma as reported in r e f 20.
HPLC 58 10-250
8 10
W h e n processing 3 mL o f
noextraction or HPLC cleanup procedures were similar, with the exception that the assay using the HPLC cleanup was about 3 times more sensitive because 3 mL instead of 1 mL of plasma was extracted (Table 111). In this regard, it should be noted that the immunoextraction filtration method could not be conveniently scaled up to handle larger plasma volumes because of the limited capacity of the GF/F filter disks to retain the immunoprecipitate without clogging. Immunoextraction centrifugation methods are under no such constraints and are therefore preferred when possible. Immunoextraction methods should be useful for the selective extraction of other low-level analytes from physiological fluids. As demonstrated here with arbaprostil and elsewhere with steroids (13-16), the selectivity of immunoextraction methods permits direct chromatographic analysis of plasma or serum extracts when it otherwise would not have been possible using conventional extraction methods. By simplification of complex sample preparation procedures, the use of immunoextraction methods can reduce time, labor, and equipment requirements. The choice between double antibody precipitation or solid-phase coupled antisera immunoextraction methods will depend on the number of samples to be processed and the supply of antiserum. Coupling an antiserum to a solid phase support may not be worthwhile for a small number of samples, but for routine applications it has the advantage that it allows the antibody to be reused. ACKNOWLEDGMENT The authors thank F. A. Bitzpatrick and M. A. Wynalda for supplying the anti-arbaprostil serum, D. P. Kane for
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supplying the goat anti-rabbit serum, R. S. P. Hsi for supplying radiolabeled arbaprostil, and M. A. Charles for assistance in preparing this manuscript. Registry No. Arbaprostil, 55028-70-1. LITERATURE CITED Goswami, S.; Mai, J.; Bruckner, G.; Kinsella, J. E. Prostaglandins 1981, 22, 693-702. Green, K.; Hamberg, M.; Samuelson, 6.; Froiich, J. C. “Advances in prostaglandin and Thromboxane Research”; Frollch, J. C., Ed.; Raven Press: New York, 1976; Vol. 5, pp 15-38. Luderer, J. R.; Riley, D. L.; Demers, L. M. J. Chromatogr. 1983, 273, 402-409. Muller, H.; Mrongovius, R.; Seyberth, H. W. J. Chromatogr. 1981, 226. 450-454. Green, K.; Hamberg, M.; Samuelson, B.; Smigel, M.; Frolich, J. C. “Advances in Prostaglandin and Thromboxane Research”; Frolich, J. C., Ed.; Raven Press: New York, 1978; Vol. 5, pp 39-94. Smith, B. J.; Heroid, D. A.; Ross, R. M.; Marquls, F. G.; Bertholf, R. L.; Ayers, C. R.; Wills, M. R.; Savory, J. Res. Commun. Chem. Pathol. Pharmacol. 1983, 40, 73-86. Dimov, V.; Green, K.; Bygdeman, M. Prostaglandins 1983, 2 5 , 225-235. Barrow, S. E.; Waddell, K. A.; Ennis, M.; Dollery, C. T.; Blair, I. A. J. Chromatogr. 1982, 239, 71-80. Christensen, P.;Green, K.; Leyssac, P. P. Acta Physiol. Scand. 1983, 117, 41-47. Reum, L.; Haustein, D.; Koolman, J. 2.Naturforsch. C : Biosci. 1981, 36C,790-797. Dyas, J.; Turkes, A,; Read, G. F.; Riad Fahmy, D. Ann. Ciin. Biochem. 1981, 18, 37-41. Glenncross, R. G.; Abeywardene, S. A.; Corney, S.J.; Morrls, H. S.J. Chromatogr. 1981, 223, 193-197. Gaskell, S.J.; Brownsey, B. G.; Groom, G. V. Ciin. Chem. (WinstonSalem, N . C . ) 1984, 30, 1696-1700. Gaskell, S. J.; Brownsey, B. G. Ciin. Chem. (Winston-Salem, N . C . ) 1983, 29, 677-680. GaskeH, S.J.; Brooks, P. W. Int. J . Mass Spectrom. Ion Phys, 1983, 48, 241-244. Gaskeil, S. J.; Brownsey, B. G.; Collins, C. J.; Lelth, H. M.; Thorne, G. C. Int. J. Mass Spectrom. Ion Phys. 1983, 46, 245-248. Royer, M. E.; KO, H., manuscript in preparation. Vantrappen, A.; Janssens, J.; Popieia, T.; Kulig, J.; Tytgat, G. N. J.; Huibregste, K.; Lambert, R.; Pauchard, J. P.; Robert, A. GastroenteroiOgy 1982, 8 3 , 357-363. Robert, A. Gastroenterology 1981, 8 0 , 1155. Pullen, R. H.; Cox, J. W. J. Chromatogr., in press. Cox, J. W.; Puiien, R. H. Anal. Chem. 1984, 56, 1866-1870. Wickremasinha, A. J.; Shaw, S. R., Thornburgh, B. A. Proceedings of the 33rd National Meeting of the Academy of Pharmaceutical Sciences, San Diego, CA, November 14-18, 1982; P-Tox p 27. Jou, Y. H.; Luo, S.C.; Bankert, R. B. J. Immunol. Methods 1983, 6 5 , 285-292. Cox, J. W.; Bothweli, W. M.; Wynalda, M. A.; Fitzpatrick, F. A,, manuscript in preparation.
RECEIVED for review April 11,1985. Accepted June 20,1985. Presented, in part, at the Second International Congress on Essential Fatty Acids and Prostaglandins, London, U.K., March 24-27, 1985.
