Anal. Chem. 1998, 70, 2191-2195
A Sample Concentrator for Sensitivity Enhancement in Chromatographic Analyses Randolph C. Galipo,† Stephen L. Morgan,† and William E. Brewer*,‡
Department of Chemistry and Biochemistry, The University of South Carolina, Columbia, South Carolina 29208, and Toxicology Department, South Carolina Law Enforcement Division, 4416 Broad River Road, Columbia, South Carolina 29210
A simple, inexpensive, fast, and sensitive method for improving detection limits using a novel sample concentrator is described. Analytes are concentrated on the tip of a GC syringe by evaporating the solvent with a flow of nitrogen. The increased sensitivity of the concentrator permits extraction with lower volumes, minimizes disposal costs, and simplifies sample preparation. The concentrator improved sensitivities for selected drugs (7.6-12.8 times), pesticides (12-17 times), and C11C16 alkanes (1.5-6 times). With or without use of the concentrator, relative standard deviations of peak areas were less than 10%. Diverse GC and GC/MS applications ranging from forensic toxicology to environmental analysis can benefit from the improved detection limits provided by this concentrator design. Preferred methods for the analysis of many environmental and toxicological samples use capillary gas chromatography/mass spectrometry (GC/MS) because of its sensitivity and specificity. These methods often require extraction of large volumes of sample and are relatively time-consuming. In some cases, these methods do not have sufficient sensitivity for analysis of ultratrace amounts of analyte or for analysis of limited volume samples. Analytical sensitivity and specificity can also be increased by use of highresolution mass spectrometry (HRMS), e.g., involving doublefocusing magnet sector instruments. When HRMS is combined with capillary GC, the determination of ultratrace compounds is possible.1,2 However, HRMS is expensive, and most laboratories do not have access to this type of instrumentation. Recently, large-volume injection has been used to improve GC/ MS sensitivity. Several approaches are available for achieving large-volume injection in capillary GC: on-column injection,3 programmed temperature vaporization injection,4-9 and splitless injection with solvent diversion.10 Large-volume on-column injections can be used to improve the chromatographic sensitivity of * To whom correspondence should be addressed: Phone: (803) 896-7385. FAX: (803) 896-7351. † The University of South Carolina. ‡ South Carolina Law Enforcement Division. (1) Cai, Z.; Ramanujam, V. M. S.; Giblin, D. E.; Gross, M. L. Anal. Chem. 1993, 65, 21-26. (2) Lopez-Avila, V.; Hirata, P.; Kraska, S.; Flanagan, M.; Taylor, J. H.; Hern, S. C. Anal. Chem. 1985, 57, 2797-2801. (3) Beltran, J.; Lopez, F. J.; Forcada, M.; Hernandez, F. Chromatographia 1997, 44, 274-278. (4) Muller, S.; Efer, J.; Engewald, W. Fresenius J. Anal. Chem. 1997, 357, 55585560. S0003-2700(97)01218-3 CCC: $15.00 Published on Web 04/08/1998
© 1998 American Chemical Society
many different types of analytes.3 Unfortunately, volatile analytes may be transferred to the analytical column during the time required to evaporate the solvent. As a result, peak splitting (double peaks) and broadening may occur, which can ultimately lead to a decrease in sensitivity. One solution to this problem is to cryogenically cool the inlet so that analytes will not be prematurely swept onto the capillary column. Hewlett-Packard (HP) recently designed an inlet system which permits large volumes (25-250 µL) to be injected into a program temperature vaporization (PTV) inlet of an HP GC (6890) while cooling the inlet.4 According to HP, large-volume injections produce lower detection limits and less thermal degradation of labile compounds. Large sample volumes are introduced to the inlet in a single injection under split flow conditions, analytes are concentrated on an adsorbent (Tenax, Carbopack) inside the PTV inlet insert, and solvent is evaporated and swept out the split vent with a rapid flow of carrier gas. The split vent is then closed after most of the solvent has evaporated, and analytes are transferred to the GC column by rapidly heating the inlet. Because of its relatively recent introduction, it is hard to evaluate performance and durability of the PTV inlet. Previous work has suggested that it is not uncommon for injection port sleeves and retention gaps to provide insufficient inertness and longevity.5 Other potential problems with this method are that chemically and thermally labile compounds may decompose at temperatures needed for desorption, salt formation may occur for aqueous injections, and analytes may break through during the enrichment process.4 Suzuki et al.10 designed a solvent diversion system with splitless injection. This method incorporates a solvent diversion column equipped with a solenoid-controlled valve. The large amount of solvent injected is passed through a cold trap column and diverted (5) Linton, C. M.; Feeney, M.; Biedermann, M. Novel Materials for Use In Large Volume Injection Capillary Gas Chromatography, Paper 397, presented at Pittcon ’97, Atlanta, GA, March 16-21, 1997. (6) Wang, H.; Wang, L.; George, J. E.; Ward, G. K.; Thoma, J. J. Analysis of Ultra-Trace Level Dioxins in Drinking Water Using Large Volume Injection GC/MS-MS, Paper 1115, presented at Pittcon ’97, Atlanta, GA, March 1621, 1997. (7) Wylie, P. L.; Wilsion, W. H.; Nixon, D. D.; Perkins, P. D. Combining Large Volume Injection with Selective GC Detection for Ultratrace Environmental Analysis, Poster 15, presented at Pittcon ’97, Atlanta, GA, March 16-21, 1997. (8) Butler, J. C.; Anderson, R.; Conoley, M.; Rankin, T. Large Volume Injections of Pesticides by ECD, Poster 380, presented at Pittcon ’97, Atlanta, GA, March 16-21, 1997. (9) Muller, S.; Efer, J.; Engewald, W. Chromatographia 1994, 38, 694-700. (10) Suzuki, T.; Yaguchi, K.; Ohnishi, K.; Yamagishi, T. J. Chromatogr. A 1994, 662, 139-146.
Analytical Chemistry, Vol. 70, No. 10, May 15, 1998 2191
Figure 1. Schematic of SCIS apparatus.
through the solvent diversion column. The valve is subsequently closed, and the analyte is eluted through the analytical column to the detector. A major drawback for large-volume injection methods is that special instrumentation is usually required, and these methods may only be available in recently purchased GC/MS instruments (for instance, the new HP 6890 GC is required for installation of the PTV inlet). In this paper, we present a simple, inexpensive, fast, and sensitive method for improving detection limits. Analytes are concentrated on the tip of a GC syringe by concentrating a plug of solvent by evaporation with a flow of nitrogen. The resultant solvent plug is then injected into a GC/MS. We refer to this device as a sample concentrator for improved sensitivity (SCIS). EXPERIMENTAL SECTION Materials. Methanol and ethanol (HPLC grade) were purchased from J. T. Baker, Inc. (Phillipsburg, NJ). Pesticide (880 ng/µL) and n-alkane (C11-C16) standard mixtures were purchased from Restek (Bellefonte, PA) and OSGE (Ringwood, Australia), respectively. Quick-Chek drug solution (Tox-Clean test mixture) was provided by Alltech (State College, PA) at a level of 50 ng/µL. Cocaine and deuterated cocaine (cocaine-d3) were provided by Sigma Chemical Co. (St. Louis, MO) and Radian International (Austin, TX), respectively. The pesticide standard mixture was diluted 10-fold with ethanol. The mixture of alkanes was diluted 400-fold with hexane. Tox-Clean standard drug mixture was diluted to 2.5, 2.0, 1.0, and 0.25 ng/µL in methanol. Calibration experiments were conducted by preparing samples containing levels of cocaine varying from 0.1 to 1.0 ng/µL (ppm) and a constant amount of deuterated standard, cocaine-d3. The area for the cocaine peak at m/z 182 was ratioed to the area for cocaine-d3 at m/z 185 and regressed against concentration. Concentrator Apparatus. A diagram of the SCIS apparatus is shown in Figure 1. The apparatus consists of a small plastic T-shaped device with a sheath inserted into one end to fit snugly around the syringe needle. The setup uses a Harvard Apparatus model 22 syringe pump (South Natick, MA), a Cole-Parmer 150mm variable area flow meter (Vernon Hills, IL), and Tygon tubing and plastic clamps (Scientific Products, McGraw Park, IL). The variable area flow meter controls the nitrogen gas flow, which is 2192 Analytical Chemistry, Vol. 70, No. 10, May 15, 1998
directed perpendicular to the syringe needle. Nitrogen flow is then directed parallel to the syringe tip to facilitate the evaporation of the solvent (Figure 1). Analytes are concentrated in a plug of solvent at the tip of a Hamilton (Reno, NV) GC syringe needle (type 2, beveled end) as the syringe pump depresses the plunger and as the gas flow evaporates solvent. Typical nitrogen flow rates and syringe pump rates are 500-4000 mL/min and 0.5-2.3 µL/ min, respectively. The concentrator apparatus was utilized under a fume hood due to the evaporation of the solvent and semivolatile compounds. GC/MS Conditions. A Hewlett-Packard (Palo Alto, CA) 5890 Series II Plus gas chromatograph coupled to an HP 5972 series mass selective detector (MSD) was used for all analyses. The GC injection port was fitted with a Merlin (Half Moon Bay, CA) microseal. Separation of analytes was performed with a 30-m × 0.25-mm DB-1 (J & W Scientific, Folsom, CA) fused silica capillary column with a film thickness of 0.25 µm. For analysis of alkanes, the oven temperature was initially held at 80 °C for 1 min and then increased to 285 °C at a rate of 12 °C/min and held at this temperature for 2 min. For analysis of the pesticide standard mixture, the oven temperature was initially held at 80 °C for 1 min then increased to 300 °C at a rate of 12 °C/min and held at this temperature for 5 min. For analysis of the Tox-Clean drug mixtures, the oven temperature was initially set at 80 °C for 4 min, ramped to 120 °C at 20 °C/min, and then increased to 290 °C at 40 °C/min and held at this temperature for 8 min. The injection port and transfer line temperatures were set at 250 and 280 °C, respectively. The mass spectrometer was scanned from 40 to 400 u for the alkane and drug standard mixtures and from 40 to 550 u for the pesticide standard mixture. All injections were made under splitless GC inlet conditions. RESULTS AND DISCUSSION Characterization of Factor Effects. Nitrogen flow rate and syringe pump rate were varied in a central composite design to characterize the dependence of the SCIS performance on these primary factors.11 The experimental design involved 12 runs at five different levels of the pump rate and nitrogen flow rate, and these factors were varied from 0.76 to 2.3 µL/min and from 480 to 4295 mL/min, respectively. Experiments were run in a random order, and the Tox-Clean test mixture was injected under each experimental condition. The integrated peak areas of the resulting chromatograms were fitted to full second-order models as a function of the two experimental factors. These fitted models for all solutes tended to show rather flat response surfaces, with associated coefficients of determination (R2 values) ranging from 0.34 to 0.67. Figure 2A shows a pseudo-three-dimensional and contour plot of the fitted response surface for desipramine (coefficient of determination, R2 ) 0.67). The null hypothesis that all factor effects are equal to zero could not be rejected (P ) 0.0701), and lack of fit of the model was not significant (P ) 0.0571). Figure 2B shows a pseudo-three-dimensional and contour plot of the fitted response surface for the sum of the integrated peak areas for all 18 drugs. This model produced an R2 value of 0.56 and exhibited no lack of fit (P ) 0.3397), and the factor effects were not different from (11) Deming, S. N.; Morgan, S. L. Experimental Design: A Chemometric Approach, 2nd ed.; Elsevier Science Publishers: New York, 1993.
Table 1. Precision of Unconcentrated (2.5 ng/µL, n ) 3) and Concentrated (1.0 ng/µL, n ) 4) Drugsa precision (% RSD) compound
unconcentrated
concentrated
amphetamine methamphetamine butabarbital amobarbital pentobarbital meperidine secobarbital glutethimide phencyclidine methadone methaqualone cocaine amitriptyline imipramine desipramine pentazocine codeine oxycodone
4.6 1.2 6.5 9.8 5.8 2.3 2.6 3.7 2.2 3.2 1.4 1.9 2.7 1.9 1.6 6.6 3.9 1.9
1.7 4.5 9.0 9.8 2.6 7.5 7.0 6.8 6.0 4.2 6.0 3.7 0.61 4.2 5.5 3.2 5.0 2.8
a For concentrated samples: syringe pump rate, 1.2 µL/min; nitrogen flow rate, 2850 mL/min; injection volume, 2 µL for all samples.
Figure 2. Response surface plots as a function of pump rate and nitrogen flow rate for (A) peak area of desipramine and (B) sum of peak areas for 18 drugs in Tox-Clean mixture.
zero (P ) 0.1829). These results demonstrate that the SCIS performance is relatively unaffected by the two experimental factors. This conclusion appears particularly valid at higher N2 flow rates. In terms of stability and reproducibility, this outcome is indicative of a robust system: the choice of operating conditions for nitrogen flow and syringe pump rate are not critical. Reproducibility. Experiments using the Tox-Clean mixture were conducted to compare precision for both concentrated and unconcentrated drugs. This drug mixture was selected because it contains drugs of different types which might provide varying degrees of recoveries with the SCIS. For example, losses of the semivolatile compounds amphetamine and methamphetamine may occur during evaporative concentration. The precision (expressed as percent relative standard deviation, %RSD) of repeated manual injections (n ) 3) was obtained for unconcentrated drugs at the 2.5 ng/µL level. The precision of repeated manual injections (n ) 4) was also obtained after the concentration of a 1.0 ng/µL standard mixture from 20 to 2 µL. The concentrator was operated at syringe pump and N2 gas flow rates of 1.2 µL/min and 2850 mL/min, respectively. The results obtained are shown in Table 1, from which it is apparent that the
Figure 3. Calibration plot for cocaine after concentration. Response is the ratio of the area for the cocaine peak at m/z 182 to the area for the cocaine-d3 standard at m/z 185 (R ) 0.994).
precision is better than 10% for both unconcentrated (1.2-9.8%) and concentrated (0.61-9.8%) standards. These results are better than those reported by Suzuki et al., who obtained relative standard deviations ranging from 3 to 16% for large-volume injections of pesticides in n-hexane.10 Hoff et al. also reported relative standard deviations between 2 and 13% for the analysis of organophosphorus pesticides by gas chromatography with large-volume on-column injection.12 Calibration. As an example of the linearity of calibration using the SCIS, Figure 3 shows a calibration plot for a representative drug component, cocaine. A straight line model (with intercept and slope parameters) fits the data with a correlation coefficient (R) of 0.994, with 5 degrees of freedom for residuals and based on experiments at five levels of drug concentration. The lack of fit of the model is not significant (P ) 0.58), indicating that a straight line relationship is adequate. Limits of Detection. Limits of detection were determined for the Tox-Clean drug components in chromatograms based on straight injections of standards and in chromatograms derived from concentrated samples. Data were acquired using full-scan (12) van der Hoff, G. R.; Baumann, R. A.; Brinkman, U. A.; van Zoonen, P. J. Chromatogr. 1993, 644, 367-373.
Analytical Chemistry, Vol. 70, No. 10, May 15, 1998
2193
Table 2. Ratios of Integrated Peak Areas for Concentrated Drugs (20 to 2 µL) to Unconcentrated Drugs peaka
name
area ratio (concd/unconcd)b
area ratio (concd/unconcd)c
detection limits (concd)d
detection limits (unconcd)e
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
amphetamine methamphetamine butabarbital amobarbital pentobarbital meperidine secobarbital glutethimide phencyclidine methadone methaqualone cocaine amitriptyline imipramine desipramine pentazocine codeine oxycodone
6.6 6.8 11.2 12.8 8.9 7.6 10.5 8.3 6.6 7.7 9.0 8.9 8.6 10.4 9.9 9.7 9.8 8.3
0.5 0.5 0.81 0.75 0.81 0.72 0.81 0.76 0.9 0.81 0.78 0.76 0.86 0.84 0.77 0.75 0.78 0.85
0.002 0.0002 0.002 0.0009 0.0008 0.0008 0.002 0.0007 0.0003 0.0002 0.0003 0.0006 0.0002 0.001 0.006 0.002 0.001 0.003
0.05 0.002 0.04 0.03 0.02 0.002 0.09 0.007 0.002 0.001 0.003 0.007 0.001 0.006 0.04 0.01 0.01 0.02
a Peak numbers correspond to Figure 4. b Area ratio determined by dividing integrated peak areas of concentrated drugs by peak areas of unconcentrated drugs (2.5 ng/µL). c Area ratios determined by dividing integrated peak areas of concentrated (20 to 2 µL) drugs at the 2.5 ng/µL level by peak areas of unconcentrated drugs (25 ng/µL). d Detection limits (ng/µL) calculated from 1.0 ng/µL level (concentrated from 20 to 2 µL). e Detection limits (ng/µL) calculated from 1.0 ng/µL level (unconcentrated).
conditions (m/z 40-400); integrated peak areas were based on extracted ions characteristic of each drug. The limit of detection was estimated by taking the ratio of the peak intensity for each component to one-fifth the peak-to-peak noise on the extracted ion chromatogram.13 Results are presented in Table 2. Even the lowest concentration of the drug mixture (0.25 ng/µL, or 250 ppm) produced integrated peak areas well above background noise (data not shown). Estimated detection limits after sample concentration are, on the average, 10-fold better than those estimated for the unconcentrated drugs. After sample concentration, all drugs are detectable at levels at or below 0.006 ng/µL, as compared to levels at or below 0.09 ng/µL without concentration. This increase of sensitivity can be extremely important for the toxicological analysis of many drugs. For example, the analysis of lysergic acid diethylamide (LSD) by GC/MS with the SCIS provides the ability to analyze this drug at levels less than 0.25 ng/mL in whole blood.14 The SCIS method for the analysis of LSD in blood is as sensitive as GC/MS using negative ion chemical ionization15 and approaches the sensitivity of the GC/MS/MS method.16 Analysis of Drugs of Abuse. For concentration studies, two scenarios were investigated. The first scenario involved injection of 2 µL of a dilute drug mixture at 2.5 ng/µL, followed by injection of the same drug mixture after concentrating from 20 to 2 µL (Figure 4). Total ion abundances and peak areas were compared to those of unconcentrated standards to determine the increase in signal achieved by sample preconcentration. Integrated peak areas for 16 drugs concentrated by our method were 7.6-12.8 times greater than the corresponding integrated peak areas of unconcentrated drugs at the 2.5 ng/µL level (with the exception of amphetamine and methamphetamine, which gave results of 6.6 and 6.8, respectively). (13) Ingle, J. D., Jr.; Crouch, S. R. Spectrochemical Analysis; Prentice-Hall: Englewood Cliffs, NJ, 1988. (14) Brewer, W. E. Improved Sensitivity for the Analysis of Lysergic Acid Diethylamide in Whole Blood by GC/MS and a Novel Concentrator. Manuscript in preparation. (15) Papac, D. I.; Foltz, R. L. J. Anal. Toxicol. 1990, 14, 189. (16) Nelson, C. C.; Foltz, R. L. Anal. Chem. 1992, 64, 1578.
2194 Analytical Chemistry, Vol. 70, No. 10, May 15, 1998
Figure 4. Total ion chromatograms of (A) the Tox-Clean drug mixture (2.5 ng/µL) and (B) the Tox-Clean drug mixture (2.5 ng/µL) after concentration from 20 to 2 µL. Concentrator conditions: syringe pump rate, 1.4 µL/min; nitrogen gas flow rate, 2554 mL/min. Peak identification shown in Table 2.
The second scenario involved comparison of peak areas after injection (2 µL) of the Tox-Clean drug mixture at the 25 ng/µL level and after injection of a 2.5 ng/µL standard (dilution factor of 10) concentrated from 20 to 2 µL. Area ratios were determined by dividing integrated peak areas of concentrated drugs by those of unconcentrated drugs (see Table 2). Theoretically, peak areas for the two chromatograms should be the same. However, smaller peak areas were observed for the mixture which had been diluted and then concentrated. With the exception of the semivolatile drugs (amphetamine and methamphetamine), the areas of the concentrated 2.5 ng/µL standard were between 0.75 and 0.90 times the areas of the unconcentrated 25 ng/µL standard. The lower results for amphetamine and methamphetamine are most probably due to their partial evaporation by the flow of nitrogen gas during the concentration procedure. It is not surprising that peak areas
Figure 5. Total ion chromatograms of (A) the unconcentrated, dilute pesticide mixture and the (B) concentrated (from 10 to 0.7 µL) dilute pesticide mixture. Concentrator conditions: syringe pump rate, 1.5 µL/min; nitrogen gas flow rate, 1780 mL/min. Peak identification: (1) 2,4,5,6-tetrachloro-m-xylene, (2) R-BHC (lindane), (3) γ-BHC, (4) heptachlor, (5) endosulfan I, (6) dieldrin, (7) endrin, (8) 4,4′-DDD, (9) 4,4′-DDT, (10) methoxychlor.
for the 25 ng/µL standard are more than 10 times that of the 2.5 ng/µL standard, since some of the drugs in the diluted standard are most probably just outside the linear dynamic range of the GC/MS analysis. Although most of the drugs are probably being preconcentrated into a “solvent plug”, some of the drugs may be adsorbed onto the needle and, therefore, not transferred to the analytical column during injection. Thus, component loss to adsorption may be the reason that the SCIS does not provide the expected 10fold increase in sensitivity. Analysis of Pesticides. Total ion chromatograms (TICs) of the pesticide mix (analytes ranging from 0.8 to 8.0 µg/mL) and concentrated (10 µL of the dilute mix concentrated to 0.7 µL) pesticides are shown in Figure 5A and B, respectively. As evident from Figure 5A, many analytes of the unconcentrated mixture were below the limits of detection. Heptachlor, dieldrin, endrin, and methoxychlor were detected, although methoxychlor was the only pesticide detected significantly above the baseline. Pesticides such as endrin (peak 7) and 4,4′-DDD (peak 8) were barely discernible from background noise. As depicted in Figure 5B, all of the analytes of the concentrated sample were easily detected. Integrated peak areas for pesticide peaks after concentration were roughly 12-17 times greater than the corresponding peak areas of pesticides in the unconcentrated mixture. Analysis of Alkanes. At some point, the vapor pressure of an analyte may be such that the effectiveness of the concentrator is limited (as noted with the semivolatiles, amphetamine and methamphetamine). We employed a mixture of alkanes dissolved in hexane to test for solute discrimination. Chromatograms of unconcentrated and concentrated (from 20 to 2 µL) alkane mixtures are shown in Figure 6A and B, respectively. The total ion abundances for the peaks from undecane to hexadecane (C11C16) did not appreciably increase upon concentration (1.5-6 times greater). Hexadecane (C16) was the only compound whose sensitivity improved significantly (6-fold increase). Peak areas of the remaining alkanes (C11-C15) only improved about 1.5 times after concentration by our method. These results suggest that com-
Figure 6. Total ion chromatograms of (A) alkane mixture diluted 400-fold and (B) alkane mixture diluted 400-fold and concentrated (from 10 to 0.7 µL). Concentrator conditions: syringe pump rate, 1.5 µL/min; nitrogen gas flow rate, 1780 mL/min. Peak identification: (1) undecane, (2) dodecane, (3) tridecane, (4) tetradecane, (5) hexadecane.
pounds with vapor pressures greater than that of hexadecane (dissolved in hexane) are apparently not good candidates for this concentration procedure. The vapor pressures of both analytes and solvents should be taken into consideration when concentrating samples. CONCLUSION The sample concentrator described here operates efficiently and robustly over a broad range of syringe pump rates and nitrogen gas flow rates. Use of the SCIS produced significantly improved sensitivities for a variety of drug and pesticide compounds. Concentration resulted in improved sensitivities for selected drugs (7.6-13-fold increase), pesticides (12-17-fold increase), and C11-C16 alkanes (1.5-6-fold increase). Relative standard deviations of peak areas for drug components before and after concentration were less than 10%, with the majority of this variability due to manual injections. The SCIS produced less than the expected 10-fold increase in sensitivity, possibly because of adsorption of analyte to the syringe needle. Future studies may investigate the use of a more inert surface to replace the syringe needle. The concentrator could be easily adapted for use with an autosampler, with more reproducible results. Because of the increased sensitivity with use of the SCIS, less sample volume may be required for extractions, sample preparation can be simplified, and disposal costs can be minimized. Diverse GC and GC/MS applications ranging from environmental chemistry to toxicology and forensic science can benefit from the improved detection limits provided by this sample concentration technique. ACKNOWLEDGMENT The authors gratefully acknowledge the support of Kenneth H. Habben (former Chief Toxicologist, South Carolina Law Enforcement Division, Columbia, SC) for this research. Received for review November 4, 1997. February 19, 1998.
Accepted
AC971218F Analytical Chemistry, Vol. 70, No. 10, May 15, 1998
2195
