Anal. Chem. 1997, 69, 1217-1222
Determination of Barbiturates by Solid-Phase Microextraction and Capillary Electrophoresis Shu Li and Stephen G. Weber*
Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
The solid phase microextraction device is based on plasticized poly(vinyl chloride) (PVC) as an extraction solvent, which is coated on a primed steel rod. The plasticizer is a phosphate triester. A simple device for back-extraction of analytes into a few microliters of backextraction solution is described. A Teflon tube, with an inside diameter just larger than the PVC-coated extraction rod, is terminated at one end by a syringe. A few microliters of back-extraction solution is placed into the open end of the tube. Because the Teflon does not wet, the drop remains together. After exposure to sample, the PVC-coated extraction rod containing the analytes is inserted into the back-extraction solution-containing tube. The aqueous back-extraction solvent spreads out in the narrow annular space thus formed. After a suitable period, the rod is removed, and the droplet re-forms. It can be ejected into a sample vial with the syringe. The device has been used in a CE-based determination of barbiturates. Extraction, back-extraction, and separation of 10 barbiturates takes less than 30 min. Extraction and preconcentration of benzoates is also briefly considered. Sample preparation is important because the matrices of biological fluids or environmental samples are so complicated that false signals are likely to be generated in typical separation-based determinations. Traditional methods of liquid-liquid extraction, although useful, will be replaced by new methods that are more efficient and that generate less organic solvent waste. Solid phase extraction (SPE)1,2 is promising because the extraction “solvent”, the stationary phase, is nonvolatile and recoverable. However, the devices are easily clogged by dirty samples, and the elution liquid, if it is volatile, needs to be evaporated to increase analyte concentration. On-line SPE-CE has gained limited success.3-5 Besides the clogging problem, injection band broadening is caused by the excessive column connections and washing procedures. Solid phase microextraction (SPME) does not have the plugging problems. It is conveniently interfaced with GC and has gained great success in environmental analysis.6-10 Recently, Chen and Pawliszyn developed an interface for HPLC11 using a tee joint in (1) Lensmeyer, G. L.; Onsager, C.; Carlson, I. H.; Wiebe, D. A. J. Chromatogr. 1995, 691, 239. (2) Galceran, M. T.; Jauregui, O. Anal. Chim. Acta 1995, 304, 75. (3) Morita, I.; Sawada, J. J. Chromatogr. 1993, 641, 375. (4) Cai, J.; Rassi, Z. E. J. Liq. Chromatogr. 1991, 559, 505. (5) Swartz, M. E.; Merion, M. J. Chromatogr. 1993, 632, 209. (6) Majors, R. E. LC-GC 1995, 13, 82. (7) Pawliszyn, J. Trends Anal. Chem. 1995, 14, 113. (8) Buchholz, K. D.; Pawliszyn, J. Anal. Chem. 1994, 66, 160. (9) Zhang, Z.; Yang, M. J.; Pawliszyn, J. Anal. Chem. 1994, 66, 845A. (10) Yang, M. J.; Pawliszyn, J. Anal. Chem. 1993, 65, 2538. S0003-2700(96)00790-1 CCC: $14.00
© 1997 American Chemical Society
the injection loop. It is probably difficult to couple SPME on-line to CE as is done with SPME-HPLC, considering that the injection volume in CE is on the order of nanoliters compared to microliters for HPLC. The purpose of sample preparation is usually twofold: eliminating interferences and concentrating the sample. Thus, selectivity and preconcentration effects can be used to evaluate the quality of sample preparation. While preconcentration may be the most important factor for clean samples, selectivity is more important in the analysis of complicated samples because noise from sample constituents is dominant over instrument noise. Ideally, an extraction system would be selective for analytes and provide preconcentration. Furthermore, the selectivity would be adjustable to accommodate the many classes of compounds that may be sought. In special cases, artificial receptors may be available to increase selectivity,12 but generally this will not be true. In general, a wide variety of solvents should be available to provide for some selectivity. We have recently studied a number of plasticizers as solvents and have discovered that tributyl phosphate is a very good solvent for barbiturates.13 While we will not achieve compound-class-selective extractions with this solvent, we will achieve organic-solvent-waste-free extraction through using plasticized poly(vinyl chloride) PVC as the extraction medium. Thus, as an intermediate step toward our goal of solvent-wastefree and selective extractions, a very simple, inexpensive extraction device for CE is reported here that operates without producing solvent waste. As it employs PVC, for which a large number of plasticizers exists, the extraction solvent is readily manipulated. In this report, we describe the physical and technical aspects of the extraction. The device is applied to the determination of barbiturates by CE.14-16 The technical questions that we have addressed concern whether we can develop a high surface areato-volume ratio extraction device based on plastisized PVC, (ii) whether we can reproducibly handle the microliter volumes of back-extraction solution, (iii) the rate of extraction, and (iv) the potential for selective preconcentration of barbiturates from aqueous buffers, human urine, and bovine serum. EXPERIMENTAL SECTION All the chemicals, unless specified otherwise, were obtained from Aldrich (Milwaukee, WI) or Sigma (St. Louis, MO). m(11) Chen, J.; Pawliszyn, J. B. Anal. Chem. 1995, 67, 2530. (12) Valenta, J. N.; Dixon, R. P.; Hamilton, A. D.; Weber, S. G. Anal. Chem. 1994, 66, 2397. (13) Valenta, J. N.; Weber, S. G. J. Chromatogr. 1996, 722, 47. (14) Thormann, W.; Meier, P.; Marcolli, C.; Binder, F. J. Chromatogr. 1991, 545, 445. (15) Meier, P.; Thormann, W. J. Chromatogr. 1991, 559, 505. (16) Shihabi, Z. K. J. Liq. Chromatogr. 1993, 16, 2059.
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Cresotinic acid and 3-methylsalicylic acid were obtained from Fluka (Ronkonkoma, NY). Solutions. Solution A contains 10 standard barbiturates in pH 4.2, 5 mM sodium acetate at the following concentrations (ppm): allobarbital 6.0, amobarbital, 5.3; aprobarbital, 6.4; butabarbital, 5.5; butalbital, 7.3; mephobarital, 1.5; pentobarbital, 6.1; phenobarbital, 7.7; secobarbital, 7.6; and thiopental, 10.1. This was diluted with sodium acetate buffer (sodium acetate 5 mM, pH 4.2) for aqueous standards. Spiked urine samples were prepared by mixing 1 part of solution A with 9 or 29 parts of a healthy human’s urine. Spiked serum samples were prepared by first mixing solution A and deionized water with volume ratios of 2/8 and 3/7. These solutions were added to the vials of lyophilized bovine serum to the specified volume. Samples were adjusted to about pH 4.5 before extraction. Acetic acid or 2 M acetate buffer (pH 4.5) was used to adjust the pH of urine samples. Spiked serum samples were adjusted to about pH 4.5 by adding 230 µL of acetic acid and 330 mg of sodium acetate to 4 mL of the spiked serum. Precipitated spiked serum was prepared by mixing 1.50 mL of acetonitrile and 1.00 mL of spiked serum. The clear supernatant was injected after centrifugation. Preparation of the Extraction Rod. Stainless steel rods (o.d. 1.1 mm, Small Parts, Miami Lakes, FL) were cut into 7 cm pieces, polished with emery cloth, cleaned with a Kimwipe and acetone, and ultrasonicated in ethanol and then tetrahydrofuran (THF) for 5 min. Poly(vinyl chloride-co-vinyl acetate-co-maleic acid) (PVAM, vinyl chloride, 86%; vinyl acetate, 13%; maleic acid, 1%) was dissolved in THF (3% w/v). The stainless steel rods were put into the THF solution of PVAM, removed from the solution immediately, held still and vertically for 1 min, and air-dried in the hood for at least 5 h. A PVAM primer is obtained on the rod. PVC (“very high molecular weight”) was added slowly to THF to 3.6% (w/v) with stirring. Santicizer 141 (2-ethylhexyl diphenyl phosphate, 92%; di-2-ethylhexyl phenyl phosphate, 5%; triphenyl phosphate, 3%; a gift from Monsanto, St. Louis, MO) was added to the PVC solution to 7.2% (v/v). The PVAM-primed rods were put into the solution, taken out immediately, held vertically for 1 min, and air-dried for at least 5 h. The length of the coating is 3 cm from one end. Rate of Extraction from Quiescent Solution. A 5 cm length of Teflon tube (i.d. 1.5 mm, Upchurch Scientific, Inc., Oak Harbor, WA) was attached to a 1 mL syringe. Fifty microliters of a standard mixture of three barbiturates (16.7 ppm thiopental, 4.2 ppm phenobarbital, and 4.2 ppm secobarbital in 5 mM sodium acetate buffer of pH 4.2) was put into the Teflon tube with a 50 µL syringe. The extraction rod was put inside the tube. The position of the rod or syringe was adjusted so that the coating was immersed in the solution. The device was left to stand horizontally for 4 min, and the rod was transferred to another identical device with fresh solution. Eight such extractions were carried out in succession with the same extraction rod. The concentrations of the three barbiturates remaining in the eight extracted solutions were determined by CE. Rate of Back-Extraction. (Refer to Figure 1). A 5 cm length of Teflon-lined heat-shrinkable tubing (i.d. 1.2 mm, Small Parts) was attached to a 1 mL syringe. The open end of the tube was enlarged slightly. Five microliters of back-extraction buffer (20 or 40 mM phosphate buffer, pH 11.5) was injected into the Teflon 1218 Analytical Chemistry, Vol. 69, No. 6, March 15, 1997
Figure 1. Device and operation. (1) Put the extraction rod in the quiescent sample solution for specific a time. (2) Inject 5 µL of backextraction solution with a 5 µL microsyringe into the Teflon tube. (3) Take out the rod from the sample solution, wipe it clean if necessary with a tissue, and put it into the Teflon tube. The droplet of solution can be forced to cover the whole surface of the membrane by adjusting the position of droplet with the syringe handle before or after placing the rod. (4) Take out the rod after letting it stand horizontally for a specific time. The majority of back-extraction solution will form a droplet near the end of the tube. (5) Collect the solution by moving the droplet spanning the diameter as a piston and transfer the drop to an injecton vial.
tube. Following extraction of analytes from the sample into the extraction rod, the rod was then put into the Teflon tube containing back-extraction buffer. After the rod was removed, the back-extraction solution was transferred to an injection vial for CE analysis. The process was repeated on the same rod with fresh back-extraction buffer until there was no analyte evident in the extract. The forward extraction was carried out from 3.5 mL of a standard mixture of 10 barbiturates (3.52 ppm each, sodium acetate 5 mM, pH 4.2). Calibration and Analysis. The device shown in Figure 1 was used. Barbiturates from the 3.5 mL quiescent aliquots of the standard solutions were extracted for 5, 10, and 30 min, and backextracted for 9, 30, and 90 min with 5 µL of phosphate buffer (20 mM, pH 11.5). Separation and Detection. Separations were carried out on an Isco 3850 capillary electropherograph (Isco Inc., Lincoln, NE). A detection window was opened 50 cm from the injection end on a 75 cm fused silica capillary (75 µm i.d., Polymicro Technologies, Inc., Phoenix, AZ). The separation buffer was 50 mM Tris adjusted to pH 7.8 with Tapso (3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid). Standards and samples were injected by applying 0.5 psi at the end of the capillary, normally for 4 s. During separation, a potential of 25 kV was applied, and the current was about 30 µA. Detection was at 230 nm. Data collection and integration were controlled by EZ Chrom (Scientific Software, Inc., San Ramon, CA). RESULTS AND DISCUSSION Device. The extractions described have used the standard SPME design of a thin extraction layer coated onto a rod. To maximize the preconcentration effect, it is necessary to perform the back-extraction with only a small solution volume. As Figure 1 shows, a device was made that forces back-extraction solution to occupy the annular space between a nonwetting tube and the
extraction rod. When the rod is removed, the majority of the liquid forms a drop spanning the tube’s diameter. Any liquid left on the wall can be collected by using the handle of the attached syringe to move this drop of liquid as a piston. More than enough back-extraction solution will be collected for CE injection. Choice of Solvent. Phosphate esters have been chosen as solvents with PVC as the matrix to hold the extraction solvent. Recently, a number of commercial plasticizers were studied for their ability to support the extraction of phenobarbital, both with and without molecular recognition.13 One plasticizer, tributyl phosphate, stood out as being a very good solvent. Similar compounds have been used in membrane-based, on-line extraction separations.17 However, for practical applications, the commonly used TBP is too water soluble. A membrane made with TPB can only be used for a single extraction. On the second use, a large decrease in the extraction efficiency is observed. Other kinds of phosphate esters have been tested. Among them, Santicizer 141 (90% octyl diphenyl phosphate) and Santicizer 148 (90% decyl diphenyl phosphate) give satisfactory results. It should be pointed out that the poor performance of TBP is not solely an issue of water solubility. It may involve interaction of water, PVC, and plasticizer in the membrane phase.18,19 The coating procedure is important for achieving uniformity and mechanical stability of membranes. Grate and McGill20 studied the wetting and dewetting phenomena of films on sensor surfaces and described a plasma cleaning method to alleviate the dewetting problem. There are also discussions on making reproducible thick- or thin-film coatings21 on electrode surfaces and automatic coating methods like screen printing or ink-jet printing.22 Plasticized PVC does not stick to stainless steel unless the steel is first coated with a primer. Poly(vinyl chloride-co-vinyl acetate-co-maleic acid) was chosen as a primer because of its good air-dry adhesion to metal.23 Operation. The preconcentration factor is defined as the ratio of the concentration of analyte in back-extraction solvent to the concentration in the sample. To increase the preconcentration factor, the extraction should be done in a pH range where barbiturates are neutral and a large partition coefficient and oilwater phase ratio would favor extraction. The pH of the backextraction should be raised high enough to ionize the barbiturates, which are weak acids, and extract them back from the oil phase to water, while the volume of aqueous phase must be minimized A pH of 11.5 has been chosen for back-extraction, which is good for barbiturates with pKa1 values of 7.3-8 and partition coefficients of 10-1000. To study the forward extraction without the bias from the backextraction, initially identical small volumes of standard were extracted multiple times with the same extraction rod. By comparison of the remaining concentration of barbiturates present in the extracted solution to standards, the quantity of barbiturates (17) Shen, Y.; Obuseng, V.; Gro ¨nburg, L.; Jo ¨nsson, J. A. J. Chromatogr. 1996, 725, 189. (18) Li, Z.; Li, X.; Petrovic, S.; Harrison, D. J. Anal. Chem. 1996, 68, 1717. (19) Sears, J. K.; Darby, J. R. The Technology of Plasticizers; John Wiley & Sons: New York, 1982; p 536. (20) Grate, J. W.; McGill, R. A. Anal. Chem. 1995, 67, 4015. (21) A-Icaza, M.; Bilitewski, U. Anal. Chem. 1993, 65, 525A. (22) Newman, J. D.; White, S. F.; Tothill, I. E.; Turners, A. P. F. Anal. Chem. 1995, 67, 4594. (23) Titow, W. V. PVC Plastics Property, Process, and Applications; Elsevier Applied Science: New York, 1990; p 684.
Figure 2. Cumulative amount of barbiturates (ng) extracted versus time. Phenobarbital (0) and secobarbital (+) use the left scale. Thiopental (O) uses the right scale.
Figure 3. Percentage of thiopental (O), phenobarbital (0), and secobarbital (+) back-extracted vs time for a 5 min extraction.
extracted can be inferred as a function of time. A single extraction is carried out every 4 min. This procedure is a discrete approximation to a continuous extraction from a large volume of solution. Figure 2 shows the quantity of extracted barbiturates versus time. The extraction power of the rod decreases with the extraction time. The fractions extracted for phenobarbital and secobarbital tend to reach a limiting value after 30 min; thus, extending the extraction time longer than 30 min will help little for the extraction of secobarbital or phenobarbital. Thiopental is unique in that the amount extracted does not go asymptotically to a constant value. This probably reflects the ability of thiopental to undergo self-association in the organic phase.24 Figure 3 shows the rates of back-extraction with 20 mM phosphate buffer of pH 11.5. Phosphate buffer of 40 mM and pH 11.5 gives the same result. For 99% back-extraction, the backextraction time is 9 min if the forward extraction time is 5 min. If the extraction time is extended to 10 and 30 min, however, the back-extraction time should be extended to 30 and 90 min, respectively. The slow back-extraction kinetics seems very likely to be of membrane origin. Calibration and Repeatability. Table 1 lists the regression results from three calibration curves (5 min extraction, 9 min backextraction). Calibration was performed twice with two different rods. The linearity is good in all cases. The variation between the different rods for the 5 min extraction is small in spite of the (24) Guerin, M.; Dumas, J. M.; Sandorfy, C. Can. J. Chem. 1980, 58, 2080.
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Table 1. Results from Regression of Concentrations of Barbiturates Injected (Calibrated by Peak Height) against Concentrations in the Corresponding Standard Solutiona secobarbital
rod1 rod2 a
thiopental
phenobarbital
R2
slope
R2
slope
R2
slope
0.996 0.999
7.5 7.6
0.996 0.999
11 13
0.996 0.999
4.3 3.2
Table 3. Preconcentration Factors for Benzoic Acids at pH 4.2 and 2.0 pH
HMBA 11a
HPAA 12
HBA 13
TA 14
CA 15
MSA 16
BA 17
ASP 18
4.2 2.0
1.4 2.4
0.5 0.9
2.2 3.5
4.8 7.2
4.1 13.3
5.1 18.9
0.3 0.6
0.3 1.1
a
The numbers following the acids match those of Figure 4.
The slope is the preconcentration factor.
Table 2. Preconcentration Factors (pf) for the Extraction of Barbiturates from Standard Solutions for Different Times and pf for the 5-min Extraction from Spiked Serum Relative to the pf from Aqueous Standards pf for standards
thiopental mephobarbitol secobarbitol amobarbitol pentobarbitol phenobarbitol butalbital butabarbitol aprobarbitol allobarbitol a
5 min
10 min
30 min
pf(std) - pf(serum) pf(std)
11.1 7.5 7.5 6.1 5.5 4.2 3.8 2.9 2.1 1.4
35.5 26.0 20.2 14.5 14.1 10.5 7.9 5.9 3.9 3.0
64.0 30.9 26.3 17.8 15.3 13.3 8.8 6.1 3.8 3.8
0.88 0.77 0.81 0.81 0.75 nda 0.70 0.75 0.57 nda
Figure 4. Separation of 10 barbiturates and eight benzoates: (1) pentobarbital, (2) butabarbital, (3) secobarbital, (4) amobarbital, (5) aprobarbital, (6) mephobarbital, (7) butalbital, (8) allobarbital, (9) thiopental, (10) phenobarbital, (11) 4-hydroxy-3-methoxybenzoic acid, (12) p-hydroxyphenylacetic acid, (13) p-hydroxybenzoic acid, (14) p-toluic acid, (15) m-cresonic acid, (16) 3-methylsalicylic acid, (17) benzoic acid, and (18) aspirin.
Quantitative analysis difficult due to interfering peak.
fact that the thickness of the coating was not strictly controlled. This is probably because the equilibrium is not established and only the outer portion of the membrane is involved for an extraction time of 5 min. The calibrations for 10 and 30 min extractions were done with only two nonzero points. All the calibrations lines pass through the origin. Table 2 shows preconcentration factors for the barbiturates with different extraction times. The relative increase in preconcentration factors is lower at longer extraction times, confirming the results shown in Figure 2 for the remainder of the barbiturates. This indicates that the membrane is far from reaching equilibrium for a 5 min extraction and that it is close to equilibrium for a 30 min extraction for most barbiturates. Since benzoates (e.g., aspirin) may be taken with barbiturates or exist as metabolites in urine, a series of benzoates was studied, too. Preconcentration factors are listed in Table 3. Separation. The Tapso-Tris buffer has a high buffering capacity but low conductivity.25 Thus, a relatively high voltage (25 kV, ∼30 µA) can be applied to improve the resolution and the speed of the separation without overheating. Figure 4 shows the separation of 10 barbiturates and eight benzoates. Compared with other buffer systems,14,15 three more barbiturates are separated in a similar length of time. Under these conditions, the benzoates and the barbiturates do not interfere with each other. Barbiturates in Urine. Blank urine and barbiturate-spiked urine were extracted for different lengths of time. Figure 5A shows that the electropherograms for the extracts (electrophero(25) Hjerten, S.; Valtcheva, L.; Elenbring, K.; Liao, J.-L. Electrophoresis 1995, 16, 584.
1220 Analytical Chemistry, Vol. 69, No. 6, March 15, 1997
Figure 5. Separation of blank and spiked urine sample. (A) Blank urine sample, (a) directly injected and extracted for (b) 5, (c) 10, and (d) 30 min. (B) Barbiturate-spiked sample, extracted for (e) 30 and (f and g) 5 min. Concentration of barbiturates in (e) and (f): (1) pento-, 0.60; (2) buta-, 0.55; (3) seco-, 0.76; (4) amo-, 0.53; (5) apro-, 0.64; (6) mepho-, 0.15; and (7) butalbital, 0.73; (9) thiopental, 1.0 ppm. Concentration in (g) is 0.3 times that of (e) or (f). (h) Blank urine, extracted for 5 min.
grams b-d) are cleaner than the electropherogram of straight urine (electropherogram a), especially where barbiturates elute (7-10 min). As the extraction time increases (b-d), the magnitudes of the peaks in the blank increase. Figure 5B shows electropherograms for barbiturate-spiked urine. A comparison of
electropherograms e and f shows that larger signals result for longer extraction times. As this is also true for the signals in the blank, there is no advantage to using long extraction times unless the electropherogram is very clean in the region of interest. This demonstrates that, even for this highly efficient separation technique, selectivity is still more important than preconcentration in prechromatographic separations of complicated biological samples. Electropherograms g, f, and h are 5 min extractions. Electropherogram h is a blank. The ratios of the peak heights of barbiturates in electropherograms g and f are proportional to the their concentration ratio (0.3:1). To establish rigorously correct detection limits, a significant number of different urine samples, taken from a cross section of society, would have to be analyzed. The statistical fluctuations in the baseline engendered by the person-to-person variations in the concentrations of compounds that elute in the 7-10 min-range would be the “noise” in the signalto noise ratio for the method. At this early stage of the development of this technique, such a labor-intensive survey is not warranted. Observations have been made on a few urine samples obtained locally (e.g., Figure 5). There are a few nonbarbiturate peaks in the region where the barbiturates elute. The signals from these peaks are less than those of 0.2 ppm barbiturates. A reasonable estimate of the lowest concentrations on which quantitative analysis can be performed is in the same range of a few tenths of a ppm. There is drift in the migration times of the barbiturates. We will discuss this below in the context of the separation in bovine serum. Barbiturates in Bovine Serum. Figure 6A shows the separation of spiked bovine serum samples treated with this method, and Figure 6B shows data for an acetonitrile precipitation. The separation of the precipitated sample (Figure 6B) uses a higher buffer concentration (100 mM Tris-Tapso) with the same pH to minimize possible sample constituent adsorption to the capillary inner surface. With this buffer, migration times are considerably longer than those with the 50 mM buffer (Figure 6A). Barbiturates in the serum sample can be detected using the extraction method (Figure 6A, electropherogram e). With the precipitation method, although the original analyte concentration is higher, no analyte signal can be detected (Figure 6B). From a quantitative analysis of the peak areas in electropherogram e, preconcentration factors can be calculated. They are uniformly lower for the serum sample than for aqueous standards. Table 2 lists the preconcentration factor in serum as a fraction of that in an aqueous standard. It is known that barbiturates bind to serum proteins,26 which will lower the extraction efficiency. Also, protein adsorption to the membrane surface during extraction will decrease the mass transport rate. To determine accurately the concentration of the barbiturates in the serum, a standard additions procedure can be used. Electropherogram a shows a sample with spikes of 0.64 ppm pentobarbital and 0.78 ppm amobarbital. The calculated values (weighed values) for the concentrations of pentobarbital and amobarbital are 1.23 (1.20) and 0.97 (1.10) ppm, respectively. In this example, the standard additions procedure is effective. Another problem that can be ameliorated by the use of standard additions is the uncertainty in compound identification (26) United States Pharmacopeial Convention. Drug Information for the Health Care Professional, USP DI. Vol. I; The United States Pharmacopeial Convention, Inc.: Rockville, MD, 1996; p 524.
Figure 6. Comparison of direct injection, extraction and precipitation methods. (A, a-e) Spiked serum treated by the extraction method and separated in 50 mM Tris-Tapso buffer. Concentrations of barbiturates before extraction in electropherograms b-e: allobarbital, 1.2; amobarbital, 1.1; aprobarbital, 1.3; butabarbitol, 1.1; butalbital, 1.46; mephobarbital, 0.3; pentobarbital, 1.2; phenobarbital 1.5; secobarbital, 1.5; and thiopental, 2.0 ppm. Electropherogram a is for the same solution as above, with additional spikes to increase pentoand amobarbital concentrations by 0.64 and 0.78 ppm, respectively. Injection: (a and e) vacuum injection, 4 s, of the extracted sample; (b-d) vacuum injection, 4 s, followed by 1 s injection of 10 ppm each of amobarbital, butabarbital, and thiopental. (B) Directly injected (f) and precipitated (g) samples and standard (h) separated in 100 mM Tris-Tapso. Concentration: (f) and (g) are 1.5 times (e); (h) is the stock solution described in the Experimental Section. Injection: (fh) vacuum injection, 4 s.
brought about by the shifting of the migration times. Certainly, the previous example shows that mixing a standard with the backextraction solution will work, but it may not be desirable to contaminate the back-extraction solution. As volumes of backextraction solution become smaller, taking an aliquot becomes more difficult. To solve this problem, vacuum injection of 4 s of the back-extraction solution have been followed by a 1 s injection of a standard. The peak with the increased height corresponds to the added standard, while the other peaks maintain their same heights. By matching the electropherogram patterns between the augmented injection and the original injection, the peaks of the latter can be identified. This is demonstrated in Figure 6A, electropherograms b-e. CONCLUSION A simple device for sample preparation based on a PVC membrane is described and applied to barbiturate determination by CE. The device is easy to construct and performs reliably. Ten barbiturates were extracted, back-extracted, and injected for Analytical Chemistry, Vol. 69, No. 6, March 15, 1997
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CE separation. Alkaline or neutral compounds are expected not to be extracted or back-extracted (respectively) and thus will not interfere with the analysis of barbiturates. Barbiturate concentrations of 0.1-0.3 ppm in urine and about 1 ppm in serum can be determined. Common benzoic acids (including aspirin) can also be extracted under the same conditions; however, they elute later than barbiturates under these separation conditions. Standard additions should be used for accurate quantitative and qualitative analysis of serum samples.
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ACKNOWLEDGMENT We are grateful to the National Science Foundation for support of this work through Grant No. CHE 9403450. We thank Monsanto for the generous gift of plasticizers, Santicizer 141 and Santicizer 148. Received for reveiew August 6, 1996. Accepted December 20, 1996.X AC960790O X
Abstract published in Advance ACS Abstracts, February 15, 1997.
