Anal. Chem. 1991, 63,819-823
819
Determination of Chlorinated Phenoxy Acid and Ester Herbicides in Soil and Water by Liquid Chromatography Particle Beam Mass Spectrometry and Ultraviolet Absorption Spectrophotometry In Suk Kim, Fassil I. Sasinos, Robert D. Stephens, Jeanny Wang, and Mark A. Brown* Hazardous Materials Laboratory, California Department of Health Services, 2151 Berkeley Way, Berkeley, California 94704
Eight chlorinated phenoxy acid and three ester herbkldes are determined in soil and water by liquid chromatography with UV absorption for quantitation and partkle beam mass spectrometry for confirmatlon. Chromatography (C-18 reversedphase column, 22 cm X 2.1 mm, water with methanol or acetonitrile and acetk acid mobile phase, 0.25 mumin flow) with UV detectbn (230 nm) gives quantitation limits of 12-80 ng in 10-pL injected volume (corresponding to 4.8-32 ppb in 125 mL of water and 20-133 ppb In 30 g of soil and 500 pL of final extract volume) with four-point c a l l k a t h ( R > 0.99). Full scan electron Ionization particle beam mass spectra are given for Chlorinated phenoxy acids at 1.25 pg each ontolunn, showing molecular and phenoxy (base) lons. Both acids and esters are efnclently and cleanly extracted from soil and water with ethyl acetate, and the esters are base hydrolyzed before analysis. The average recovery of elght carboxylic acids spiked into water at 33.3, 1.0, and 0.1 ppm and spiked into soil at 33.3 and 1.0 ppm is 78% (average standard deviation 4.2%). The average recovery and ester hydrolysis efficiency for the three esters 2,4-D mixed Isobutyl, 2,4,5-T butoxyethanoi ether, and 2,4-DB Isobutyl spiked at the same levels In soil and water is 88% (average standard deviation 10%).
INTRODUCTION The determination of many important polar target environmental pollutants by conventional gas chromatography (GC) methods requires derivatization to less polar and more volatile adducts. A standard method for the analysis of the chlorinated phenoxy acids and their corresponding ester herbicides in soil and water specifies extraction with diethyl ether, alkaline hydrolysis, and (re)esterification via diazomethane. Separation of the methyl esters is accomplished with GC, quantitation with an electron capture (or other) detector, and confirmation with GC/mass spectrometry (I). Although diazomethane is both explosive and toxic, the methylation step is required to make it possible to resolve these analytes with a conventional GC column. Liquid chromatographic (LC) separation is not restricted to more volatile analytes and therefore is suitable for the separation of free carboxylic acids without prior derivatization. The separation of chlorinated phenoxy acid herbicides by reversed-phase liquid chromatography has been reported with photoconductivity (2)and UV absorption (3) detection. Because of the legal implications of environmental analytical data, the analyte confirmation provided by full scan electron ionization MS detection is an important aspect of any environmental method. The confirmation of an analyte may be
* To whom correspondence should be addressed.
as important as its quantitation. T h e GC/MS method described above for chlorinated phenoxy acids and esters provides an excellent confirmation based on the numerous and highly reproducible diagnostic fragmentation ions produced in the electron ionization (EI) mode. A LC method should ideally provide the same level analyte confirmation confidence. Thus, although thermospray LC/MS is useful for the analysis of environmental pollutants including chlorinated phenoxy acid herbicides (4,the very soft ionization provided by this method yields very few ion fragments for analyte confirmation. This problem is partially circumvented by techniques including the use of solvent adduct ions with the use of special solvents (5) and with collision-activated dissociation tandem mass spectrometry experiments to produce further fragmentation of the major ions produced by the thermospray ionization process (6). The development of particle beam as a commercial LC/MS interface giving E1 spectra creates the possibility of a simple LC/MS method for the direct analysis of chlorinated phenoxy acid herbicides with the confirmatory power of the corresponding GC/MS method. Particle beam electron ionization MS has an advantage in this sense over thermospray because it provides conventional E1 spectra that can be compared to standard reference spectra. This is possible in part because E1 spectra are largely independent of spectrometer conditions. Liquid chromatography particle beam mass spectrometry has been successfully used for the determination of other target and nontarget nonvolatile pollutants in aqueous leachates (7). This paper describes the extraction with ethyl acetate of chlorinated phenoxy acid and ester herbicides from soil and water, hydrolysis to the acid, separation by reversed-phase chromatography, and quantitation by UV absorption and confirmation by particle beam mass spectrometry detection.
EXPERIMENTAL SECTION Reagents and Chemicals. Authentic chlorinated phenoxy acid and ester herbicides and one chlorinated benzoic acid herbicide are obtained from the U.S.EPA Pesticides and Industrial Chemicals Repository (U.S.EPA, Research Triangle Park, NC) and include the following compounds (Table I) (purity >99%): 2-methoxy-3,6-dichlorobenzoicacid (Dicamba); 2,4-dichlorophenoxyacetic acid (2,4-D);4-chlorc-2-methylphenoxyaceticacid (MCPA); 2,4,5-trichlorophenoxyaceticacid (2,4,5-T); 2-(2,4-dich1orophenoxy)propionic acid (2,4-DP); 2-(4-chloro-2-methylphen0xy)propionic acid (MCPP); 2-(2,4,5-trichlorophenoxy)propionic acid (2,4,5-TP); 4-(2,4-dichlorophenoxy)butyricacid (2,4DB); and 4-(4-chlorc-2-methylphenoxy)butyric acid (MCPB). The “mixed isobutyl”, butoxyethanol ether, and isobutyl esters of 2,4-D, 2,4,5-T, and 2,4-DB are obtained from the same source. Liquid Chromatography Apparatus and Conditions. Liquid chromatography uses a Hewlett-Packard 1050 liquid chromatograph equipped with a 1040 diode array detector and 79994A ‘Chem Station” for data acquisition. Reversed-phase chromatography columns, obtained from Brownlee Labs, Applied Biosystems Inc. (Santa Clara, CA), are 2.1-mm i.d. and either 10
0003-2700/91/0363-0819$02.50/00 1991 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 8, APRIL 15, 1991
Table I. Liquid Chromatography Retention Times of Eight Chlorophenoxy Acid Herbicides and Dicamba, Mobile Solvent Conditions, Calibration Correlation Coefficients (R), and Corresponding Quantitation Limits (QL)" (UV Absorbance Detection) retention time. min analyte (free acid)
10-cm col gradient
Dicamba 2,4-D MCPA 2,4,5-T 2,4-DP MCPP 2,4,5-TP 2,4-DB MCPB
4.5
10.2
7.4
14.8 16.5 18.5 19.6
22-cm col gradient isocratic
7.6 9.9 9.5 9.6
21.5 24.8
11.7 11.1
39.2
NA
42.1
NA 12.6 13.3 21.8
20.3 21.1
36.7 28.8 30.9
4-pt calib (22-cm col) R QL, ng NA 0.995 0.999 0.992 0.991 0.992 0.997 0.997 0.992
NA 120 24
148 149 98 90 105 124
Quantitation limit is the concentration of the linear 4-point calibration line corresponding to the lower 95% prediction limit n intercept, Le., the minimum concentration at which the predicted Deak area includes zero at the 95% confidence limit. Table 11. Reversed-Phase Liquid Chromatography Gradient Solvent Conditions for the 10- and 22-cm Reversed-Phase Columnsa % mobile-phase comp
time, min 0 2
8 14
water
acetonitrile
acetic acid (20% aq)
10-cm Column 65 30 60 35 50 45 0 95
5 5 5
5
90 mobile-phase comp
time, min 0
10
water
methanol
22-cm Column 55 10 10 55
acetic acid (1% aq)* 35 35
a Isocratic conditions with the 22-cm column were water (59%), acetonitrile (36%), and acetic acid (20% aqueous; 5%). *With 10 mM ammonium acetate.
or 22 cm in length (ODs-102 Spheri-5 and ODS-222 Spheri-5, respectively). Reversed-phase liquid chromatography isocratic and gradient solvent conditions are shown in Table 11. Injections of more dilute solutions with volumes greater than 10 pL use an initial low methanol or acetonitrile concentration in the mobile phase of 5%, followed by a rapid gradient (1 min) to the initial solvent composition shown in Table 11. Calibration and Quantitation w i t h UV Absorption Detection. Calibration for quantitation is based on four-point calibration curves with triplicate on-column injections (10 pL) a t 10,30,60, and 100 ppm each of a mixture of eight chlorinated phenoxy acid herbicide standards (Table I) and a solvent blank. Quantitation is a t 230 nm employing peak areas for integration. Daily calibration uses triplicate injections a t 50 ppm and is considered within an acceptable range if a response is within &15% of the expected value based on the original four-point calibration curve. Soil Samples. Soil samples are obtained from samples submitted to the Hazardous Materials Laboratory, California Department of Health Services, that had been determined by a standard GC method to contain less than 0.5 ppm chlorinated phenoxy acids. Spiking Soil and Water Samples. Soil and distilled water samples are spiked with chlorinated phenoxy acid or ester analytical standards (mixture of 1000 ng/pL each standard in ace-
tone). Soil (10 or 30 g) is spiked with the solution of standards (333 or 30 rL) to give spike levels of 33.3 or 1.0 ppm, respectively. Distilled water (10,30, or 125 mL) is spiked with the solution of standards (333, 30, or 12.5 pL) to give spike levels of 33.3, 1.0, and 0.1 ppm, respectively. Extraction of Chlorinated Phenoxy Acids and Esters from Soil or Water. With soil samples, distilled water (20 mL) is added and the mixture mechanically shaken for 5 min a t room temperature. Water samples, or the resulting aqueous fraction of soil samples, are acidified (pH 2) with hydrochloric acid (2 M) before extraction. Chlorinated phenoxycarboxylic acids or esters are extracted from the acidified aqueous mixture with three portions of ethyl acetate (30 mL each). The combined extracts are centrifuged for 5 min at 2000g, the aqueous (bottom) layer is removed with a glass pipet, and the remaining organic layer is dried over anhydrous sodium sulfate (2 g) and decanted. Ethyl acetate (10 mL) and ethyl alcohol (5 mL) are used sequentially to wash the residual sodium sulfate. The combined extracts and washes are evaporated under vacuum with a rotary evaporator. If the soil or water sample is spiked exclusively with the chlorinated phenoxy-free carboxylic acids, i.e., no esters present, the residue is transferred with four portions of methanol (1 mL each) to a vial. The combined methanolic solution is dried on a sand bath under a gentle nitrogen flow a t 40 "C. The residue is dissolved in methanol (250 pL) for quantitative analysis. Hydrolysis of Chlorinated Phenoxy Acid Esters. If the soil or water sample is spiked with chlorinated phenoxy acid esters, then the residue obtained after removal of ethyl acetate with the rotary evaporator is dissolved in a solution of ethanol (20 mL) and aqueous KOH (37%,5 mL). This mixture is placed in a water bath a t 65 "C for 1h and then cooled to room temperature. The pH of the solution is adjusted to 2 with hydrochloric acid (2 M) and extracted with three portions of ethyl acetate (30 mL each) in a separatory funnel. The combined extracts are centrifuged for 5 min a t 2000g, the aqueous (bottom) layer is removed, and the organic layer is dried over anhydrous sodium sulfate and decanted into a 250-mL round-bottomed flask. The solvent is removed under vacuum with a rotary evaporator and the residue transferred to a vial in methanol (250 pL) as described above. Particle Beam Mass Spectrometry Apparatus. Instrumentation consists of a Hewlett-Packard 5988A mass spectrometer equipped with a Hewlett-Packard particle beam liquid chromatograph interface and 1090 high-performance liquid chromatograph (Hewlett-Packard, Palo Alto, CA). Particle beam mass spectrometry full scan mode (from 62 to 400 amu) with electron ionization uses a source temperature of 250 "C, an electron multiplier voltage of 1650-2900 V, a scan cycle time of approximately 2 s, a particle beam interface helium flow of 50 psi, and temperature of 45 "C. The nebulizer setting is optimized with caffeine as a standard as described by the manufacturer. Mass spectra are produced by summing two to four scans across a chromatographic peak followed by background subtraction of an equal number of scans taken immediately before that peak.
RESULTS A N D D I S C U S S I O N Efforts t o use particle beam mass spectrometry for quantitation as well as confirmation were not satisfactory. In our hands, the instrument response factors of the particle beam mass spectrometer to the chlorinated phenoxy acid standards varied by as much as 2-fold in a 24-h period. Reasonable accuracy required daily recalibration over the relevant concentration range. Therefore, we took advantage of the much more reproducible UV absorption chromatography for quantitation. Reversed-Phase Chromatography: UV Absorption Detection. Table I1 shows the retention times for eight chlorinated phenoxy acid herbicides under gradient conditions with the 10- and 22-cm columns and under isocratic conditions with the 22-cm column. Table I1 also shows t h e correlation coefficients and quantitation limits for calibration with the 22-cm column, with isocratic solvent conditions and UV absorption detection. Correlation coefficients range from 0.991 t o 0.999 with quantitation limits between 24 and 149 ng, corresponding to 2.4-14.9 ppm in the final extract/concentrate
ANALYTICAL CHEMISTRY, VOL. 63, NO. 8, APRIL 15, 1991
821
Chlorophenoxy
s
Acid Standards
sY CI
1 ; 4
8
T
200 I
141
I 12 16 20 24 28 32 36 4 0 Min dl
1J
Flgure 3. Electron ionization mass spectra comparing (a, top) chlorophenoxy acid herbicides 2,443 and MCPA from the chromatographic peaks in Figure 2 (1.25 pg of each analyte injected in 10 ML) and (b, bottom)the corresponding NBS-Wlley llbrary mass condensed spectra (not all peaks shown).
I
4
8
12
16
20
24
2 0 32
36
40Min
Flgure 1. UV absorbance chromatogram (230 nm) showing (a, top) the separation of eight chlorophenoxy acid standards and Dicamba at 200 ppm injected (2 pg in 10 pL) and (b, bottom) the extract of soil spiked at 33.3 ppm with eight chlorophenoxy acid herbicides. Chromatography is with the 22-cm column; gradient conditions (Table 11).
Ir
I.
'b121i
s.,g
M
1W
120 140
0
100
M
1W 120 140 1M IC4 200 220 240 260
1M l M
200 220 244 2M
M
100
110 140
IW
IM 200 220
ZM
Chlorophenoxy
n
Acid Standards
120
140
IM 200
160
220
240
0 I W 120 140 lM 180 ?m 220 240 260
Spiked Soil
Extract
1,
,
Flgure 2. Total ion particle beam mass spectrometry chromatogram
(electron ionization) showing (a, top) the separation of eight chlorophenoxy acid standards and Dicamba at 125 ppm injected (1.25 pg in 10 pL) and (b, bottom) the extract of soil spiked at 33.3 with eight chlorophenoxy acid herbicides. Chromatography is with the 22-cm column: gradient conditions (Table 11). (10-pL injection volume) under these conditions. A typical
UV absorption chromatogram (230 nm) showing the separation profile a t 200 ppm injected (2.0 Kg in 10 p L ) is shown in Figure la.
Reversed-Phase Chromatography: Particle Beam Mass Spectrometry Confirmation. A typical electron ionization total ion chromatogram showing the separation profile a t 125 ppm injected (1.25 pg in 10 pL) is shown in Figure 2a. Figure 3 shows a comparison of representative particle beam mass spectra (the first two eluting chlorophenoxy acid herbicides 2,4-D and MCPA) to the corresponding NBS-Wiley reference library mass spectra (Hewlett-Packard). The reference spectra are in a condensed form and do not show every ion present in the original spectra. Comparison shows that the spectra are visually very similar. All the ions present in the reference spectra are also present in the particle beam spectra, but the relative intensities are somewhat different. We have observed that the relative intensity of the parent molecular ion in the particle beam spectra
,,:
,?",\,,
,
,I, M
,I, I/, , !
100
120
140
,
IM
,
,
IM
,
,
'r".,
200
220
Flgure 4. Electron ionization mass spectra for chlorophenoxy acid herbicides corresponding to chromatographic peaks 3-8 in Figure 2 (1.25 pg of each analyte injected in 10 pL). is invariably smaller than that seen in the corresponding library spectra. The E1 mass spectra of the next six eluting chlorinated phenoxy acid herbicides are shown in Figure 4. All spectra show an appreciable parent ion with appropriate C1 isotope cluster. The base ion for each spectrum, as in conventional E1 spectrometry, is the corresponding phenoxy ion, as seen in Figure 3. The phenoxy base ion, seen for the E1 spectra of all the chlorinated phenoxy acid herbicides, corresponds to the loss of the corresponding alkanecarboxylic acid moiety and rearrangement with transfer of a proton to produce the corresponding phenol rather than the phenolate ion. In each case, the identity of the peak is unambiguously confirmed by inspection of its mass spectrum, although a detectable carryover or "memory" effect is also seen. For example, the spectrum of MCPP (Figure 4) shows, in addition to a good parent ion and phenoxy ion a t m / z 214 and 142, smaller ions at m/z 234,220,200, and 162 that clearly remain from earlier eluting analytes. Possibly the low volatility of these compounds relative to the corresponding methyl esters that would be used with GC/MS causes them to clear from the ion source more slowly.
822
I
ANALYTICAL CHEMISTRY, VOL. 63, NO. 8, APRIL 15, 1991
Table IV. Recovery and Alkaline Hydrolysis Efficiency of Three Chlorophenoxy Acid Esters Spiked into Water and Soil spike recovery and ester hydrolysis efficiency, % f SD"
analyte (ester)
a
61
a U
2,4-D mixed
water spike, ppm 33.3
1.0
soil spike, ppm 0.1
1 .o
33.3
108 f 15 91 f 1.9 72 f 11 106 f 13 115 f 4.9
isobutyl esters 2,4,5-T butoxy-
95 f 15 84 f 11
72 f 15
75 f 8.2
93
12
ethanol ether
I 4
6
8
'
..I"
1
10
'
1
'
I
'
12 Min.
jb
Figure 5 . Total ion current chromatograms (a, top) of seven chloro-
phenoxy acid herbicides and Dicamba (2.0 pg each, 1000 ppm) with the short 10-cm column, gradient conditions, and the extracted ion profiles (lower traces) of the corresponding phenoxy fragment ions. The total ion chromatogram (b, bottom) of the same compounds of 0.2 pg each, 10 ppm is also shown under these conditions (Table 11). Table 111. Recovery Efficiency of Eight Chlorophenoxy Acid Herbicides Spiked into Water and Soil analyte (free acid) 2,4-D MCPA 2,4,5-T 2,4-DP MCPP 2,4,5-TP 2,4-DB MCPB
spike recovery, '70 f SD" water spike, ppm soil spike, ppm
33.3
1.0
0.1
*
78 f 1.0 76 f 4.5 77 2.6 77 f 0.6 69 f 4.1 74 f 1.5 83 f 1.5 110 f 11 80 f 5.4 78 f 2.0 71 f 4.0 79 f 5.9 81 f 3.8 69 f 2.5 74 f 2.9 89 f 1.2 84 + 7.9 74 9.0 81 f 5.6 17 f 3.8 73 f 3.3 85 f 1.7 82 f 6.3 84 f 6.1
*
33.3
*
82 3.1 75 f 1.5 87 f 3.6 80 f 3.2 79 f 4.5 85 f 2 . 1 82 f 1.5 81 f 2.1
1.0 79 f 5.9 70 f 6.2 71 f 4.7 77 f 9.9 72 f 3.0 69 f 6.0 77 f 14 68 f 7.2
" Standard deviation based on 3 replicates. E x t r a c t e d I o n Profiles: More Rapid Determination of Coeluting P e a k s on a S h o r t (10-cm) Column. With a simpler mixture of chlorinated phenoxy acid herbicides, a faster, isocratic chromatographic system with a shorter column is useful. The added dimension of data from particle beam mass spectrometry detection allows the unambiguous identification of partially coeluting peaks seen under these conditions. Figure 5 shows the total ion current chromatograms (a) of seven of the chlorinated phenoxy analytes and Dicamba. The extracted ion profiles (lower traces) of the corresponding phenoxy (base) fragment ions allow the resolution and confirmation of individual analytes contained in each peak. Detection limits under these conditions are approximately 10 ppm, i.e., the lowest standard concentration injected (Figure 5b, 200 ng in 20 pL). EPA method 8150 for chlorinated phenoxy acid herbicides also includes Dalapon and Dinoseb as target analytes (I). With the method described above, only the chlorinated phenoxy acid and ester herbicides and Dicamba are detected. At 2 pg per injection, neither Dalapon (probably too volatile) or Dinoseb is detected. Poor response of particle beam due to dinitrophenols has been previously observed (8). Recovery Efficiency of Eight Chlorinated PhenoxyFree Acids Spiked i n Water a n d Soil. Table I11 shows the recovery efficiency for eight chlorinated phenoxy acid herbicides in water at 33.3, 1.0, and 0.1 ppm and soil at 33.3 and
esters 2,4-DB isobutyl
9 4 f 17 86 f 4.9 71 f 18
66
* 5.0
89 f 5.1
ester a
Standard deviation based on 3 replicates.
1.0 ppm. Distilled water is not an ideal model for all types of actual aqueous environmental samples, and these recovery data and detection limits correspond to a minimum matrix interference situation. Reversed-phase chromatography with UV absorbance and mass spectrometry detection of extracts of soil spiked at 33.3 ppm are shown in Figures 1 and 2 (b traces), respectively. The use of ethyl acetate as an extraction solvent for the free acids and the esters in soil appears to give reasonable selectivity, i.e., good efficiency of extraction coupled with minimal extraction of interferences such as humic acids (Figures 1 and 2, b traces). This solvent offers increased safety in use compared to diethyl ether, which is specified in a standard EPA method (I). Alkaline Hydrolysis p l u s Recovery Efficiency of T h r e e Chlorinated Phenoxy Acid Esters Spiked in Water a n d Soil. Although many different esters of the chlorinated phenoxy acids have been produced by various manufacturers, alkaline hydrolysis of the extract from soil or water reduces the number of analytes to the eight free carboxylic acids. Table IV shows the efficiency of recovery and alkaline hydrolysis to the free carboxylic acid for three chlorinated phenoxy acid esters spiked in water a t 33.3,1.0, and 0.1 ppm and soil at 33.3 and 1.0 ppm. The three esters are chosen to represent the alcohol moieties commonly esterified to chlorinated phenoxy acid herbicides by different herbicide manufacturers. These include the mixed isobutyl, isobutyl, and butoxyethanol ether esters (nomenclature from the supplier of the standards (I)). Reversed-phase chromatography electron ionization particle beam mass spectrometry provides a sensitive and useful alternative to GC/MS based methods for the analysis of chlorinated phenoxy acid and ester herbicides. It has the advantage of not requiring (re)esterification of the acid herbicides and is capable of directly analyzing the acids with only a single chromatography column. Registry No. MCPA, 94-74-6; 2,4,5-T, 93-76-5; 2,4-DP, 12036-5; MCPP, 708519-0; 2,4,5-TP, 93-72-1; 2,4-DB, 94-82-6; MCPB, 94-81-5; Dicamba, 1918-00-9; water, 7732-18-5.
LITERATURE C I T E D Environmental Protection Agency-Test Methods for Evaluating SoM Waste (SW-846). 3rd ed.; Offlce of sdid Waste and Emergency Response, U.S. Government Printing Office: Washington, DC, 1986. Miles, C. J.; Zhou, M. J . A@. Food Chem. 1990, 38, 986-989. Di Corcia, A.; Marchetti, M.; Samperi, R. Anal. Chem. 1989, 67, 1363- 1367. Voyksner, R. D. In Applications of New Mass Spectrometry Techniques in Pesticide Chemistry; Rosen, J. D., Ed.; John Wiley and Sons: New York, 1987; p 146. Barcelo, D. In Liquid ChromatographylMass Spectrometry: AppHcetion in Agricufiural, pharmaceutical and Envkonmental Chemistry; Brown, M. A,, Ed.; ACS Symposium Series 420; American Chemical
Anal. Chem. 1991, 63, 823-827
Society: Washington, DC, Jones, T. L.; Betowski, L.
1990; pp 48-61. D.; Yinon, J. In Li9u/d Chromtographyl Mess Spectrometry: Apphtkm In Agvfcuthual, pharmaceutical and Envkmmental Chemistry; Brown, M. A., Ed.; ACS Symposium Series 420; American Chemical Society: Washington, DC, 1990; pp 62-74. (7) Brown, M. A.; Kim, I . S.; Sasinos, F. I.; Stephens, R. D. Environ. Scl. Technol. 1990, 24, 1832-1636. (8) Brown, M. A.; Kim, I. S.; Sasinos, F. I.; Stephens, R. D. In Li9uM ChromatographylMass Spectrometry: Application in Agricultural,
(6)
823
pharmaceutical and E n v k m m t a l Chemistry; Brown, M. A., Ed.; ACS Symposium Series 420 American Chemical Society: Washington, DC, 1990; pp 198-214.
R E C E ~for D review October 15, 1990. Accepted January 7, lggl*M. A*Brown Qatefu1b' financial SUPPofi from the U.S. EPA EMSL, Las Vegas, NV.
Micellar Electrokinetic Capillary Chromatography of Illicit Drug Substances Robert Weinberger*-' Applied Biosystems, Inc., 170 Williams Drive, Ramsey, New Jersey 07446
Ira S.Lurie
Drug Enforcement Administration, Special Testing and Research Laboratory, 7704 Old Springhouse Road, McLean, Virginia 221 02
Micellar electrokinetic capillary chromatography (MECC) was found to give slgnlfkantly greater efficiency, selectivity, peak symmetry, and speed compared to high-performance liquid chromatography (HPLC) for the determination of llllcit drug substances. For a complex mixture consisting of acldlc and neutral Impurities present In an illicit heroin seizure sample, MECC resolved at least twice as many peaks as HPLC. MECC permitted the analysis of heroin and Its basic impurities, the common adulterants phenobarbital and methaqualone, In approximately one-third the analysis time of HPLC with superior resolution. IlHcIt cocaine, and Its basic impurities, were analyzed by MECC without the significant tailing that Is found with reversed-phase liquid chromatography (LC) using bonded-phase columns. Other drugs investigated via MECC include opium alkaloids, amphetamines, hallucinogens, barbiturates, benzodiazepines, and cannablnoids. All of these separations were accompkhed with 25100-cm caplliarles (length to detector) by using a hydroorganic buffer consisting of 85 mM sodium dodecyl sulfate, 8.5 mM phosphate, 8.5 mM borate and 15% acetonitrile at a pH of 8.5. Detection was by uttravioiet (UV) absorption at 210 nm. Due to its speed, high resolving power, and the probability that all compounds must elute at or before t, (micellar aggregate mlgration tlme), MECC is well suited for general drug screening.
INTRODUCTION Illicit drug substances invariably consist of compounds that are polar, thermally degradable, or nonvolatile and thus can be difficult to analyze via gas chromatography (GC). Highperformance liquid chromatography (HPLC) has been employed (1-4) but in general lacks the resolving power of capillary GC (5, 6). Forensic drug substances that are clandestinely manufactured such as heroin, methamphetamine, Current address: CE Technologies, P.O. Box 140,Chappaqua,
NY 10514.
0003-2700/91/0363-0823$02.50/0
and fentanyl can be highly complex. For example a heroin sample can consist of heroin, its basic, acidic, and neutral manufacturing impurities, and various adulterants. Natural products such as opium and psilocybin contain exogenous plant and processing matrix impurities. In still other examples, matrix complications such as dyed blotter paper or parsley are characteristic of the dosage forms for LSD and PCP. For a myriad of forensic drug samples HPLC affords adequate resolution but gives fair to poor peak shape for many basic compounds. Many of these drug determinations require gradient elution LC to speed the analysis yet the time of separation can still be quite long ( 4 ) . Micellar electrokinetic capillary chromatography (MECC) is a subclass of capillary electrophoresis (CE). First described by Terabe et al. in 1984 (7, 8), the technique brings reversed-phase and ion-pairing mechanisms to CE as additional tools in developing separations. MECC provides the opportunity to separate in a single run, both neutral and charged molecules in an electroosmotically driven system, an application that was not possible before its inception. A great variety of molecular types are amenable to MECC including phenols (7),chlorinated phenols (9,101,phenylthiohydantoin amino acids ( I l ) , nucleosides and oligonucleotides (12),nucleic acids (13),catechols (14),vitamins (15,16), antibiotics (17), chiral substances (18,19), isotopic substituents (20),peptides (21,22),barbiturates (22),and porphyrins (23). The goal of this paper is to assess the applicability of MECC for the separation of illicit drugs. Comparisons are made with HPLC separations currently being employed in forensic laboratories.
EXPERIMENTAL SECTION Instrumentation. A Model 270A capillary electrophoresis system (Applied Biosystems, Inc., San Jose, CA) was employed for all MECC studies. A chromatography data system (Model 7500 Perkin-Elmer, Norwalk, CT) or ChromJet integrator (SpectraPhysics, San Jose, CA) was used for data handling. A prototype fluorescence detector adaptation to the CE instrument was previously described (23). Four different sized capillaries (Polymicro Technologies, Scottsdale, AZ) were used: 122 cm X 50 gm i.d., 72 cm X 50 pm 0 1991 American Chemical Society
