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Anal. Chem. 1989, 61, 540-544
Preparation and Certification of Standard Reference Material 1507: 11-Nor-Ag-tetrahydrocannabinol-9-carboxylic Acid in Freeze-Dried Urine Neal E. Craft,* G a r y D. Byrd,' a n d L a u r e n c e R. Hilpert2
Center for Analytical Chemistry, National Institute of Standards and Technology (formerly National Bureau of Standards), Gaithersburg, Maryland 20899
The National Institute of Standards and Technology (NIST) has prepared and certlfled SRM 1507, a freeze-dried urine f o r t W wlth 1lnor-AO-tetrahydrocannabkroC9-carboxyllc ackl (THC-9-COOH), the malor urinary metabolite of marlJuana. The certified concentration of 20 f 1 ng/mL for the anaiyte was obtained from the concordant results of analyses of the materlal by gas chromatography/mass spectrometry (GWMS) and high-performance tiquld Chromatography with electrochemical detection (HPLC-EC). Soild-phase extractlon was used to prepare the sample for GC/MS analyses, and iiquldliquid extraction was used for the HPLC-EC analyses. The multistep HPLC method was developed at NIST to ckcunvenl Interferences from urlnary constituents. The rewlts of a round roMn test on this material among five Department of Defense laboratories Involved In drug testing are reported.
INTRODUCTION The growth and awareness of illegal drug use in our society has stimulated widespread testing for substance abuse. Quality assurance in drug testing is a critical feature in the overall process of identifying and treating drug abuse. Testing positive for an illegal drug carries severe consequences in many cases. Because of the economic and social consequences involved, measurements for drugs or drug metabolites in physiological fluids must be accurate at the low-nanogramper-milliliter level. Urine samples collected for testing purposes are currently screened by immunoassays, and positive responses are confirmed by a more specific and legally admissible technique such as gas chromatography/mass spectrometry (GC/MS). Analytical methods for testing for drugs of abuse, such as marijuana, have appeared in the literature in recent years (1-10). Independent of the method used, reliable control samples such as Standard Reference Materials (SRM's) can be useful tools for improving the quality and ensuring the accuracy of measurements in analytical laboratories. An SRM is a well-characterized material containing the analytes of interest at certified concentrations. At the National Institute of Standards and Technology, three modes of measurement have historically been used to obtain accurate analyte concentrations during certification. These are the use of (a) definitive methods, (b) reference methods, and (c) two or more independent and reliable methods. The separate SRM units are produced from a large batch of material, and after packaging, the finished lot is checked for homogeneity. In an effort to provide reliable standards for the drug testing community, the NIST has prepared and certified SRM 1507,
* Author to whom correspondence should be addressed.
Current address: R. J. Reynolds-Nabisco Co., Bowman Gray Technical Center, Winston-Salem, NC 27102. Current address: Central Coast Analytical Services, Inc., San Luis Obispo, CA 93401.
a freeze-dried urine fortified with 11-nor-Ag-tetrahydrocannabinol-9-carboxylic acid (THC-9-COOH),the major urinary metabolite of marijuana. This is the first of a series of urine matrix S R M s being produced containing drugs of abuse. Each unit of SRM 1507 consists of three bottles of freeze-dried urine: two bottles containing THC-9-COOH at the certified concentration of 20 i 1 ng/mL (after reconstitution) and one bottle of a urine blank. The certified value for the analyte was obtained from the concordant results of measurements by two independent methods. The use of two totally independent methods minimizes the probability that any bias associated with one method will be duplicated in the second. The first method utilized solid-phase extraction to isolate the THC-9-COOH from the urine, followed by GC/MS measurement. The second method involved liquid-liquid extraction to isolate the THC-9-COOH, followed by high-performance liquid chromatography (HPLC) analysis with electrochemical (EC) detection. This paper describes the preparation and certification of a unique material, SRM 1507. We also present the results of an interlaboratory comparison of the measurement of THC-9-COOH in SRM 1507 conducted among several Department of Defense laboratories involved in drug testing. EXPERIMENTAL SECTION Chemicals. Neat THC-9-COOH and an ethanolic solution of the isotopically labeled internal standard 11-nor- Ag-tetrahydrocannabinol-5'-d3-9-carboxylicacid (THC-d3-9-COOH)were obtained from Research Triangle Institute (Research Triangle Park, NC). Analysis of the THC-9-COOHat NIST by direct probe mass spectrometry and GC/MS of the trimethylsilyl (TMS) derivative showed the purity to be greater than 99%. The internal standard used for the HPLC measurements, 11-nor-11-hydroxy-A9-tetrahydrocannabinol (THC-11-OH), was obtained as an ethanolic solution from Walter Reed Army Medical Center (Washington, DC). Solvents used for HPLC measurements were of HPLC grade, and all other chemicals were of reagent grade or better and were used without further purification. Apparatus. CISSep-Pak solid-phaseextraction cartridges were obtained from Waters Associates (Milford, MA). The Mixxor solvent extraction system was obtained from Lidex Technologies, Inc. (Bedford, MA). The GC/MS analyses were carried out with a gas chromatograph interfaced to a quadrupole mass spectrometer (HewlettPackard Company, Palo Alto, CA). Chromatographic separations were carried out on a 30 m X 0.25 mmDB-5 fused silica capillary column (J&W Scientific, Rancho Cordova, CA), which was interfaced directly to the ion source of the mass spectrometer. The GC had a split flow injection port, which was maintained at a temperature of 275 "C. Helium was used as the carrier gas with a head pressure of 100 kPa (15 psi), and the split flow ratio was 20:l. The temperature of the column was initially held at 225 O C for 2 min and then programmed to 277 "C at a rate of 4 OC/min. The column was then heated ballistically to 300 "C and held for 2 min to clear the column of late-eluting material. The mass spectrometer was operated in the electron impact mode with an ionizing energy of 70 eV and an ion source temperature of 200
This article not subject to US. Copyright. Published 1989 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 61, NO. 6, MARCH 15, 1989
"C. The selected ion monitoring mode was used to improve the selectivity and sensitivity of detection. Ions a t m/z 488 ( M + for the trimethylsilyl derivative of THC-9-COOH) and at m / z 491 (M'+ for the trimethylsilyl derivative of THC-d3-9-COOH) were monitored with a dwell time of 150 ms, which ensured that at least 20 data points were obtained across each chromatographic peak. The HPLC analyses were performed with a solvent delivery system consisting of two single-piston pumps, a gradient controller, and a high-pressure gradient mixer (Beckman Instrument Company, San Ramon, CA) with an added pulse damper. The chromatographic separation was accomplished by using a 5-pm Ultrasphere C18 analytical column (0.46 X 25 cm) protected by a C18guard column (Rainin Instrument Company, Woburn, MA). A model 400 EC detector (EG&G Princeton Applied Research, Lawenceville, NJ) fitted with a glassy carbon working electrode and a Ag/AgCl reference electrode was used in the oxidative mode a t an applied potential of +lo00 mV. The injection volume was 30 pL, and the flow rate was 1.6 mL/min. A multistep gradient program with two premixed solvents was necessary to provide adequate resolution of the THC-9-COOH and the internal standard from naturally occurring interferences in the urine extracts. Solvent A consisted of 54% water, 36% acetonitrile, 10% methanol, and 1.3 mL/L glacial acetic acid; solvent B was 27% water, 63% acetonitrile, 10% methanol, and 1.15 mL/L glacial acetic acid. The f i t step of the solvent program was a steep linear gradient from 5% B to 70% B in 15 min to elute the majority of polar materials in the urine extracts. Next, a shallow linear gradient from 70% B to 76% B over 10 rnin was incorporated to provide a level base line during the elution of the THC-11-OH and THC-9-COOH. This was followed by a steep linear gradient to 100% B in 3 min, which was held for 5 min to wash late-eluting material from the column. The column was then returned to the initial conditions and equilibrated for 10 min prior to the next injection. Collection and Preparation of Material. The urine used to prepare SRM 1507 was voluntarily donated by NIST staff members. It was assumed that this population would provide a low blank urine with minimal viral contamination. Individual samples were collected in 250-mL polypropylene specimen cups and transferred to two 50-L polypropylene carboys. The urine was chilled in an ice bath during the 2 days of sample collection. Samples of bulk urine were hydrolyzed with glucuronidase and analyzed for the presence of THC metabolites prior to further processing; no THC metabolites were found. Processing of the urine for SRM 1507, including aseptic filtering, bottling, and lyophilization, was carried out under sterile conditions a t BellMore Labs, Hampstead, MD. The bulk urine was processed in two lots: the first lot (approximately 30 L) was the urine blank; the second lot (approximately 50 L) was fortified with THC-9COOH. Each lot of urine was filtered through a 0.45-pm cellulose acetate filter. To the second lot of filtered urine, 1.3 mg of THC-9-COOH in 10 mL of ethanol was added. The fortified and blank urine samples were each homogenized for approximately one-half hour by swirling, allowed to stand for 1h, and then mixed by swirling for an additional 15 min immediately prior to bottling. The blank and fortified urine samples were dispensed into precleaned 50-mL amber glass bottles (25 mL per bottle). To check the fill volume reproducibility, we determined the net weight of urine added to each of 11bottles selected at random over the entire filling sequence. The net weight of urine added to each bottle varied by less than 0.5% relative standard deviation. Loosely stoppered bottles of urine were frozen a t -35 "C and then lyophilized a t 50-85 Torr for 48 h a t 0 "C and an additional 48 h a t 40 "C. The lyophilized SRM is stored a t 6 "C. Sample Selection for Certification. Although both blank and fortified urine samples were analyzed, only the THC-9-COOH concentration in the fortified urine was certified. Twenty-four samples representing each of the 24 trays of lyophilized urine were selected a t random from the fortified urine and analyzed in duplicate by GC/MS. They were extracted in random order and measured in two batches on separate days. Ten HPLC measurements of THC-9-COOH were made by using seven randomly selected urine samples representing the entire lyophilization process. Sample Reconstitution. Each sample selected for analysis was reconstituted by adding 25.0 mL of HPLC grade water with
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a calibrated volumetric pipet. The sample bottle was recapped with the original rubber stopper, and its contents were mixed by swirling and gently inverting the sample several times. The sample was then allowed to sit for 30 min with occasional swirling to ensure complete dissolution of all of the material. Solid-Phase Extraction and GC/MS Analysis. Two aliquots (10 mL each) were removed from each bottle for analysis. Each 10-mL aliquot was placed in a 50-mL centrifuge tube and processed to isolate the THC-9-COOH from urine by using a modification of a published procedure ( I ) . To each 10-mL urine sample, 200 ng of the isotopically labeled internal standard THC-d3-9-COOH in 100 pL of ethanol was added immediately after the sample was removed from the bottle. The urine samples were acidified to pH 3-4 by adding 250 pL of glacial acetic acid. Each sample was loaded onto a C18 cartridge at a rate of 5-10 mL/min. The C18was conditioned prior to loading the sample by wetting with 3 mL of methanol followed by 5 mL of water. The CI8 cartridge was washed with 5 mL of water followed by 5 mL of 40% (v/v) acetonitrile in water. The THC-9-COOH was eluted from the cartridge with 3 mL of methanol. The eluent was evaporated to dryness under a gentle stream of nitrogen, and the residue was dissolved in 100 pL of N,O-bis(trimethylsily1)acetamide (BSA) and heated a t 50 "C for 15 rnin to convert the THC-9-COOH to the bis(trimethylsily1) (TMS) derivative for GC/MS analysis. The chromatographic peaks in the urine extracts corresponding to the analyte and internal standard were identified by comparing retention times with solutions of standards. Calibration standards, made from independent gravimetrically prepared solutions of THC-9-COOH and the THC-d3-9-COOH solution, were analyzed to determine the response factor for the THC-9-COOH relative to the internal standard. The urine extracts were analyzed by GC/MS in groups of six to nine samples. Three independently prepared calibration standards were analyzed by GC/MS once prior to and once following each group of urine extracts. An average response factor for each group of urine extracts was calculated from six analyses of the calibration standards. The amount of THC-9-COOH in the urine samples was calculated from the ratio of peak areas for the analyte and the internal standard, using the average response factor. A BSA sample blank was analyzed following each set of calibration standards and after each set of urine extracts to check for any sample carryover. None was found. Figure 1 shows selected ion chromatograms for m / z 488 (THC-9-COOH TMS, Cz7H,04Siz) and m / z 491 (THC-d3-9-COOH TMS, C27H41D304Si2) from analysis of a calibration standard, an extract from a fortified urine sample, and an extract from a blank urine sample. Liquid-Liquid Extraction and HPLC Analysis. The following extraction method and HPLC analysis were developed at NIST and used in the certification of SRM 1507. Each aliquot (10 mL) of reconstituted urine was placed into the chamber of a Mixxor apparatus, and 200 ng of THC-11-OH was added in 100 pL of ethanol. The pH was adjusted to 3-4 with 250 pL of glacial acetic acid, and the sample was extracted twice with 15 mL of 3% (v/v) isobutyl alcohol in hexane. The organic phase was passed through no. 40 fiter paper and dried under a gentle stream of nitrogen a t 50 "C. The residue was transferred to a concentration tube with three 1-ML portions of methanol and concentrated to 100 pL. Calibration of the HPLC system was performed by using a fixed volume of the THC-11-OH internal standard solution and three independent gravimetrically prepared standards containing concentrations of THC-9-COOH in methanol which encompass the SRM concentration. Calibration standards were initially measured in triplicate to establish the response factor and the linearity of the calibration solutions. Response factors determined on the following days of analysis did not alter the initial average by more than 1.5%. For that reason, daily response factors were averaged with the previously established response factor. Peak heights were measured electronically, but each chromatogram was visually evaluated and reintegrated as necessary. Representative chromatograms of a calibration standard, a fortified urine extract, and a blank urine extract are presented in Figure 2.
RESULTS AND DISCUSSION GC/MS is the most commonly used method to confirm the presence of drugs and their metabolites in urine (2) and was
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Figure 1. Selected ion chromatogramsof m l z 488 and m l z 491 for (a) calibration standard, (b) extract of fortified urine sample, and (c) extract of blank urine sample. employed for the determination of THC-9-COOH in this study. Gravimetric values could not be used as a second measurement approach due to adsorption and partitioning of the THC-9-COOH. Therefore, HPLC was selected as an independent measurement approach to confirm the GC/MS results for the certification of SRM 1507. The HPLC method used was developed at NIST when several published methods (3-6) failed to resolve satisfactorily the THC-9-COOH or the internal standard from the urinary components present in the SRM material. During initial attempts to establish an HPLC method, the Ae isomer of THC-g-COOH, 11-nor-As-tetrahydrocannabinol-9-carboxylicacid, was investigated as the internal standard. Unfortunately, SRM 1507 contains a dominant interfering compound that elutes very close to the analyte and is both UV-absorbing and electroactive. I t was extremely difficult to separate the THC-9-COOH and, in particular, the As isomer from this interference. THC-11-OH, which was more easily resolved from the interference, was therefore used as the internal standard. For routine drug testing this would be an inappropriate internal standard since THC-11-OH is a metabolite of THC. However, in the certification of this SRM it was ideal, since the compounds possess similar chemistry and electroactivity and no native THC-11-OH was found. Isocratic elution, which eliminates the problem of detector base-line drift at high sensitivity, was tried but failed to resolve the analytes sufficiently from the interfering urinary constituents. I t was necessary to incorporate a multistep gradient to accomplish this separation which maintained a steady base line during analyte elution.
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Figure 2. Representative HPLC-EC chromatogramsof (a) Calibration standard, (b) extract of fortified urine sample, and (c) extract of blank urine sample. Different amounts of acetic acid were added to solvents A and B to offset the UV absorbance of the organic solvents. Although adequate signal was observed with UV detection, superior selectivity and sensitivity were achieved with electrochemical detection as shown in Figure 3. This observation has been reported by others ( 4 , 5 ) . In light of the multitude of interfering compounds in individual urine samples and the decreased selectivity of both UV and EC detection relative to that of MS, this HPLC method is not recommended for confirmation of marijuana abuse. T o keep the two analytical methods used as independent as possible, the urine samples measured by GC/MS were processed by using solid-phase extraction, and those measured by HPLC-EC were processed with liquid-liquid extraction. Multiple standards were independently prepared for each technique, and unrelated instrumental measurements (GC/ MS and HPLC-EC) were used to determine the concentration of THC-9-COOH in the urine extracts. Results from the GC/MS and HPLC analyses of the THC-9-COOH concentration in SRM 1507 as well as the certified value for the THC-9-COOH concentration are shown in Table I. Data from the two measurement approaches used for the certification of SRM 1507 were in good agreement. The GC/MS assay of the THC-9-COOH concentration in SRM 1507 gave an average value of 19.9 ng/mL with the standard deviation of a single measurement being 0.6 ng/mL for 48 measurements used in the certification. The HPLC assay gave a value of 19.7 ng/mL with the standard deviation of a single measurement being 0.4 ng/mL for 10 measurements. A round robin analysis of this material was conducted among five Department of Defense laboratories that use
ANALYTICAL CHEMISTRY, VOL. 61, NO. 6, MARCH 15, 1989
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Time (minutes) Flgure 3. Comparison of (a) HPLC-EC and (b) HPLC-UV chromatograms of an extract of a fortified urine sample.
Table I. Concentration of THC-9-COOH in SRM 1507 analytical method
concn, ng/mLa
GC/MS HPLC-EC certified concentrationb
19.9 f 0.6 (n = 48) 19.7 f 0.4 (n = 10) 20 f 1 c
Mean f SD of a single measurement. Certified concentration is based on the concordant results obtained from two independent analytical procedures. The listed estimated uncertainty is based on a statistical evaluation of random errors plus an allowance for possible systematic bias. Subsequent measurements of SRM 1507 by HPLC-EC and GC/MS one year after the initial certification have resulted in a new certified concentration, 20 f 2 ng/mL. GC/MS for routine drug testing. The average concentration reported from those laboratories was 20.8 ng/mL with the mean standard deviation among the laboratories being 1.0 ng/mL. The THC-9-COOH concentrations of individual aliquots from the samples reported by those laboratories ranged from 17.7 to 22.0 ng/mL. Based on our experience with interlaboratory round robin studies on the determination of trace level constituents in complex mixtures, these numbers are in good agreement with the NIST certified value of 20 f 1 ng/mL. During preparation of the fortified urine batch, the THC-9-COOH was added at a concentration of approximately 26 ng/mL. Preliminary work in our laboratory on smaller batches of urine had indicated that some of the THC-9-COOH is lost during the bottling and lyophilization processes. Thus, it was not considered unusual that about 25% of the added material was not recovered. While this prohibited the use of any gravimetric data for certifying the SRM, the use of both independent extraction and chromatographic analysis provided us with the two independent procedures needed to establish the certified concentration. None of the bottled material was discarded at the beginning or end of the filling process so that the homogeneity of the entire lot could be assessed. Analyses were performed on bottles from several trays, specifically including the first and
543
last trays to see if the bottling procedure was reproducible in delivering the same concentration of analyte throughout the process. The results indicated that some of the bottles in tray 1 contained unusually low concentrations of THC9-COOH (as low as 16.6 ng/mL), which may have been caused by nonequilibrium within the dispensing apparatus a t the start. Tray 27 contained only 15 bottles, which were the last to be filled, and some of these showed unusually high concentrations of THC-9-COOH (ashigh as 40 ng/mL). This was believed to be the result of analyte enrichment in the urine foam as discussed below. Due to the anomalous results obtained from samples in the first and last trays, trays 1, 26, and 27 were not included as part of the certified SRM lot. The higher concentrations of THC-9-COOH measured in the final tray were of particular interest. The urine was drawn out of the carboy from the bottom, and the batch was not stirred during the bottling step. Significant agitation of urine such as that which preceded the bottling step will produce foam at the top. Even though the urine was filtered, some particulate matter remained suspended in the urine and may be concentrated in the foam. It is possible that THC-9-COOH could adsorb onto this particulate matter to produce samples enriched in THC-9-COOH which were bottled with the last available portion of the batch. For this reason, the reconstituted samples were swirled to mix instead of being shaken. The latter process will produce significant amounts of foam and possible inhomogeneity in the sample. Preliminary work on a small batch of freeze-dried urine fortified with THC-9-COOH showed that no significant loss of analyte occurred over the course of 1 year after the lyophilization process. Therefore we anticipate that the THC-9-COOH concentration in SRM 1507 will be stable in its lyophilized form for at least 1 year under refrigeration. SRM 1507 will be monitored continually at NIST for any indications of degradation. The blank urine that is included as part of SRM 1507 is not certified for the concentration of THC-9-COOH. However, several samples from the reconstituted blank urine were extracted by the two procedures and analyzed. No THC-9-COOH was detected in any of the blank urine samples. The limit of detection for the THC-9-COOH is less than 1 ng/mL when the procedures described above are used. SRM 1507 is the first of a series of standard reference materials for drugs of abuse. The measurement approaches used are not necessarily intended to be routine, cost effective, or time effective but demonstrate the accuracy and precision with which such measurements can be made. ACKNOWLEDGMENT We wish to thank Robert C. Paule for his statistical consultation during this project. Registry No. THC-9-COOH, 56354-06-4. LITERATURE CITED (1) Nakamura, G. R.; Stall, W. J.; Masters, R. G.; Folen, V. A. Anal. Chem. 1985, 5 7 , 1494-1496. (2) Schwartz, R. H.; Hawkes, R . L. J . Am. Med. Assoc. 1985, 254, 788-792. (3) El Sohiy, M. A.; El Sohly, H. N.;Jones, A. B.; Dimson, P. A,; Wells, K. E. J . Anal. Toxicol. 1983, 7 , 262-264. (4) Nakahara, Y.; Sekine, H. J . Anal. Toxicol. 1985, 9 , 121-124. (5) Isenschmid, D. S . ; Caplan, Y. H. J . Anal. Toxicoi. 1986, IO, 170-1 74. (6) Szepesy, L.; Horvdth, M.; Szdntb, J.; Veress, T. Extraction and HPLC Analysis of Cannabinoids. In Chromatography ' 8 4 ; Kaldsz, H.; Ettre, L. S., Eds.; Akaddmiai Kiadb: Budapest, Hungary, 1986. (7) Thompson, L. K.; Cone, E. J. J . Chromarogr. 1987, 421, 91-97. (S) Evans, M. A.; Morarity, T. J . Anal. Toxicoi. 1980, 4 , 19-22. (9) Ayyanger, N. R.; Bhide, S . R. J . Chromarogr. 1986, 366, 435-438. (10) Lurie, I. S.; Carr, S. M. J . L i q . Chromatogr. 1986, 9 , 2485-2509.
RECEIVED for review June 17,1988. Resubmitted November 28,1988. Accepted December 15,1988. Partial support for
Anal. Chem. 1989, 6 1 , 544-547
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development of this SRM was provided by the office of the Assistant Secretary for Health Affairs, U.S. Department of Defense. Certain commercial equipment, instruments, or materials are identified in this paper to specify adequately
the experimental procedure. Such identification does not imply recommendation or endorsement by NIST, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.
Shipboard Determination of Aluminum in Seawater at the Nanomolar Level by Electron Capture Detection Gas Chromatography C. I. Measures* and J. M. Edmond Department of Earth, Atmospheric a n d Planetary Sciences, E34-246, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
A method for the determination of Ai in seawater at levels between 0.6 and 120 nM is described. The technique uses electron capture detection of the l,l,l-trifiuoro-2,4-pentanedione derivative of Ai, whkh is prepared by solvent extraction from a 15-mL sample of seawater. The method, which has a detection iimH of 0.6 nM and a relative precidon of f3.8% at 18.5 nM, has been used at sea on eight oceanographic cruises.
Trace elements with short residence times in the Ocean are, when coupled with strong input functions, nontransient tracers of their injection process. Aluminum is such an element. Despite its being the third most abundant element ( 1 ) in the earth's crust (8.2% by weight), dissolved aluminium concentrations in seawater are reported to fall between 0.2 ( 2 ) and 120 nM (3) in the trace element range. These low concentrations are a result of the low solubility of aluminosilicate phases during continental weathering ( 4 ) and the short residence time of the dissolved form in the ocean. The distribution of dissolved A1 in profiles from the Pacific and Atlantic leads to the conclusion that the dominant process in the delivery of A1 to the marine environment is the partial dissolution of atmospherically derived aluminosilicate dusts in the surface waters of the oceans (5). The short residence time of the element in the dissolved form ensures that background concentrations remain low, allowing the imprint of this input function to remain sharply defined. Shore-based determination of trace elements from samples collected a t sea is the modus operandi for most applications; the ability to acquire precise and accurate data rapidly under the adverse conditions that exist onboard ship is clearly of benefit. Not only does this provide the opportunity to modify sampling strategies as data gathering proceeds, but also provides the ability to identify and solve any unforeseen contamination problems in the field. The technique presented here is an evolution of the method originally presented for the determination of Be in seawater ( 6 ) . While the methods use mutually exclusive handling protocols and different solvents, they are both part of a continuing program that is aimed at adapting and developing analytical techniques for trace elements that exploit the high sensitivity and seagoing capability of electron capture detection gas chromatography. While many techniques for the determination of aluminum in freshwaters have been developed (7-9), only two techniques have been applied successfully to the determination of the element in seawater where the concentration levels are sig0003-2700/89/0361-0544$01 S O / O
nificantly lower. The fluorometric lumogallion method of Hydes and Liss ( I O ) , which has a detection limit of ca. 2 nM and a precision of 5% a t 37 nM, has been used successfully both at sea and in the laboratory on stored samples by a variety of workers to produce high-quality data in the Atlantic and other oceans (11-15). Orians and Bruland ( 2 ) have achieved the best detection limits of ca 0.1 nmol/kg in a shore-based analytical scheme by solvent extraction of 250-g samples of previously frozen Pacific Ocean water with 8hydroxyquinoline in a class 100 clean room and then by using atomic absorption spectroscopy to quantify the concentrated Al.
EXPERIMENTAL SECTION Apparatus. A Hewlett-Packard 5792 gas chromatograph (ECD-GC) equipped with a 10-15-mCi 63Ni electron capture detector was used for all determinations. The ECD-GC was run in the split mode (split ratio approx. 401) with a 15 m X 0.3 mm 0.d. 0.2-pm film DB 210 capillary column (J&W Scientific). The detector temperature was maintained at 350 OC;the injection port and column were heated to 250 and 130 "C, respectively. The pressure at the top of the column was set at 15 psi, equivalent to a linear velocity of about 45 cm/s. The column gas was hydrogen (Matheson zero grade), which had been passed through a 13X molecular sieve trap (HP no. 5060-9084). Detector makeup gas (95% argon, 5% methane) was also cleaned by using a 13X molecular sieve and an oxygen trap (Matheson no. 6406) and supplied at 45 mL/min. Distillation of the l,l,l-trifluoro-2,4pentanedione (Htfa, Eastman Kodak) has been described earlier (6). Small-volume separatory funnels are not commercially available; the use of the smallest ones available (125 mL) results in a considerable fraction of the solvent being left behind on the walls of the vessel. A simple low-volume (ca. 20 mL) Teflon separatory funnel can be constructed from an approximately 18 cm X 1.5 cm piece of heat shrink Teflon tubing shrunk onto a standard Teflon seperatory funnel stopcock. Sample shaking was performed with a Burrel Model 75 wrist action shaker. Reagents and Standards. The buffer, 1M sodium acetate, is prepared from twice recrystallized material; all of the commercially available grades were found to contain unacceptably high levels of Al. The recrystallization procedure is as follows: 87 g of NaAe3H20 is dissolved in 67 mL of sub-boiled distilled water in a 250-mL Teflon bottle. The solution is filtered through an 0.45-pM Nuclepore filter held in a previously acid leached filtration unit (Millipore). To this solution is added 100 mL of absolute ethanol, which has been redistilled in the Teflon microstill used for the Htfa purification. After the precipitate has formed (overnight in a refrigerator), the material is filtered in the same manner as above. The once recrystallized material is dried under a filtered stream of air at room temperature, and a second recrystallization performed, as above. The yield at each step is approximately 50%. 1989 American Chemical Society
