Analyses of Fish Tissue by Vacuum Distillation/Gas Chromatography/Mass Spectrometry

Table of Contents

INTRODUCTION
Fish tissue is an important medium for study due to its importance as a food source and as an indicator of the quality of the environment.¹ The application of vacuum distillation/gas chromatography/mass spectrometry (VD/GC/MS) for tissue analyses has been reported for volatile organic compounds (VOCs)² and is being promulgated as a Resource Conservation and Recovery Act (RCRA) method (SW 846 Method 5032).³ It has been shown that surrogate-based matrix corrections can improve method performance by accurately measuring and compensating for matrix effects from water, soil, and oil.^4,5 This work was undertaken to apply the surrogate-based matrix corrections to expand the Method 5032 analyte list and to obtain lower method detection limits for analytes in fish tissue.

Before the vacuum distillation of analytes from fish tissue could be evaluated, it was necessary to ensure added compounds were in equilibrium with the matrix. Three means of adding anaytes to the fish tissue were investigated. Determining the interval of time necessary for a spike to equilibrate required an assumption. The assumption was that an analyte was at equilibrium with the matrix when its recovery from the matrix did not decline with further equilibration time. The surrogate-based matrix correction data were generated using samples of fish tissue equilibrated with the spiked analytes.

The bioaccumulation of organic compounds for fish is closely related to the octanol-water partition coefficients.⁶ Therefore a desirable matrix correction treatment should emphasize the accuracy for analytes with higher octanol-water partition coefficients. Conversely, for those analytes that have lower octanol-water partition coefficients, water becomes a better indicator of their concentration in the environment and their presence in fish tissue are not likely due to environmental exposure.

The reported surrogate-based matrix correction model requires the solution of an equation relating analyte recovery to two factors, its partitioning between a sample and the vapor phase (water-air partition coefficient), and its condensation in the apparatus (corresponding to boiling point). The values of constants in the equation are determined using the recovery of surrogate compounds. The recovery of analytes from a tissue sample by vacuum distillation was expected to be described using this equation as it had been shown to describe the recovery of analytes from oil.⁵

A second model with a factor describing the vaporization of analytes from the organic phase became necessary when samples larger than 1 g were analyzed. It was discovered that the vacuum distillation of analytes from an organic matrix corresponds closely to the compound's K_oa and is the primary factor for analytes with higher K_ow.

Both models required the use of relative volatility (a comparison of water-air partition coefficients for different compounds) as the means of addressing matrix effects relating to the partitioning of analytes between the vapor phase and the water or organic phase. While relative volatility (a) is described as a ratio of two values (K_wa) it is convenient to assign values for relative volatility that correspond to their partition coefficients.⁵ In this work the relative volatility of an analyte from the organic phase into the vapor phase (a_Koa) is assigned values that correspond to the octanol-air partition coefficient (K_oa). The a_Koa values used in this study were determined using the experimental technique reported for the determination of the a_Kw.⁵

EXPERIMENTAL SECTION

Vacuum Distillation Apparatus. The vacuum distiller has been previously described.⁵ In the current study, a Nupro toggle valve (0.172-in. orifice) was used as the sample chamber valve. A pirani vacuum (Edwards Model 1000) gauge placed at the vacuum pump to monitor the integrity of the apparatus under vacuum. The sample chamber six port valve temperature was maintained at 150 °C (Valcon E rotor). The cryoloop was modified by using nichrome wrapping for heating which replaced the need for a hot water bath.

The vacuum distiller was modified to allow a helium sweep of the condenser column to remove condensate between vacuum distillations. A helium transfer line (5 psi) was connected at the top of the condenser column (Nupro toggle valve) and a helium exit vent (Nupro toggle valve) was attached to the transfer line between the sample chamber valve and the condenser.

The condenser column was normally held at 5-10 °C during vacuum distillations and at 60 °C between distillations. Water was used to replace isopropyl alcohol as the temperature-controlling fluid in the condenser.

Vacuum Spike Apparatus. A series of valves (Nupro toggle 4BKT) was connected to a 1/4 in. manifold. The manifold was also connected to a vacuum pump (Edwards E2M-1) and a thermocouple vacuum gauge. The valves and manifold were wrapped with heating tape and heated to 60 °C during use. The valves were connected to 15 mm O-ring fittings made of stainless steel for connection to the glass sample vessels also used for vacuum distillation.

GC/MS Apparatus. A Hewlett-Packard mass spectrometer (Model 5972) and gas chromatograph (HP5890 Series II with Model MJSC metal jet separator) with a 60-m x 0.53-mm i.d., 3.0-um film thickness, VOCOL capillary column (Supelco, Bellefonte, PA) was used for the determination of analytes from the vacuum distillation apparatus. Gas chromatograph operating conditions were: 3 min at 10 °C, 50 °C/min ramp to 40 °C; 5 °C/min ramp to 120 °C; 20 °C/min ramp to 220 °C; and isothermal at 220 °C for 3.4 min, resulting in a total run time of 28 min. The jet separator was held at 210 °C and the transfer line held at 280 °C. The mass spectrometer was operated at 3.1-s scans of 38 to 270 amu. The injector was interfaced to the vacuum distillation apparatus by connecting the carrier inlet gas line to the cryoloop valve and back to the injector. The injection or inlet temperature was 240 °C and the inlet pressure was 10 psi.

Sample Preparation. Samples were prepared by mixing analytes with 1 to 10 g aliquots of fish tissue. Canned tuna (packed in water) was used as the matrix studied for method development. Seven different fish composites were obtained from EPA Region 9 (San Fransico, CA) and were used to evaluate the method performance for 1 g aliquots. Five mL water was added to the samples prepared for water spike or sonication spike investigations. One mL water was added to samples to assist mixing after completion of the equilibration studies.

Tissue samples were spiked with analytes dissolved in 10 µL methanol directly in sample vessels, (a 100-mL round bottom flask fitted with a 15 mm O-ring connector ) that were also used to contain the sample during vacuum distillation. Analytes and their concentrations used for this study are listed in Table 1.  Additional surrogates were added for this study and their relative volatilities were experimentally determined using the same technique as reported for the other analytes.⁵ The additional surrogates and their assigned relative volatility values are nitromethane-d₃ 510 ± 260; tetrahydrofuran-d₈, 355 ± 11; nitrobenzene-d₅, 87.5 ± 33.5; and diethyl ether-d₁₀, 32.5 ± 3.0. Tetrahydrofuran-d₈ was used in this study as a replacement for acetone-d₆ as acetone-d₆ underwent rapid deuterium-hydrogen exchange in the tissue matrix.

Vacuum Distillation Procedure. Prior to a vacuum distillation, the condenser column was cooled to 5 °C to condense water being evaporated from the sample. The sample chamber containing the sample (room temperature) was evacuated for 5 min. Most water vapors were collected on the condenser column, and the distillate, containing the analytes to be transferred to the GC/MS, collected in the cryoloop immersed in liquid nitrogen (-196 °C). The sample chamber valve was closed at the completion of the vacuum distillation and the cryoloop valve switched to allow the GC carrier gas to sweep the cryoloop. The cryoloop's liquid nitrogen bath was removed and the cryoloop temperature was ballistically heated to 120 °C to volatilize the distillate. After the transfer of the distillate to the GC was complete (3 min), the cryoloop was heated to 200 °C and then allowed to cool to room temperature. After the sample was vacuum distilled, the condenser column was heated to 60 °C, the condenser flushed with helium, while the helium/condenser valve and vent valve were opened to remove most of the trapped material. After 3 min the condenser-helium line and vent valves were closed and the system was evacuated with the vacuum pump for an additional 10 min period to remove any condensed water and potential contaminants remaining after the He flush.

Spike Procedures. The vacuum spike was performed on 1 and 10 g aliquots of tissue. A sample chamber containing the tissue and analyte spike (no water added) was attached to the vacuum spiking apparatus and emersed in a liquid nitrogen bath (-196 °C). After 5 min of emersion in the cryogenic bath the tissue was completely frozen. The sample chamber was then evacuated by slowly opening the vacuum valve (between the manifold and sample chamber) and leaving it open until the pressure in the chamber was 1 torr. The sample was then isolated by closing the valve and the sample was warmed to room temperature (approximately 3 min when emersed in a warm water bath). After the sample thawed the equilibration period began. At the completion of the equilibration time water (1 or 5 mL) was added and the sample was immediately analyzed.

The water spikes were performed simply by adding the spike solution to the 1 g tuna 5 mL water matrix, the sample swirled, and allowed to stand. O-ring connector caps (TG Scientific Glass, Irvine, CA) with Viton O-rings sealed the samples during their equilibratioN period performed at room temperature (18 to 22 °C).

The sonication spike was prepared like the water spike but with the sample sonicated during the equilibration period. A Cole-Parmer, Model 08855-00 ultrasonic cleaner (Niles, Illinois) was modified to allow tap water to flow through the tank maintaining a 15-17 °C bath during continuous operation. The sample chamber was emersed in an ultrasonic water bath to a depth where the sample is just below the surface of the water.

RESULTS AND DISCUSSION

Before any surrogate-correction model could be evaluated, it was necessary to ensure the analytes added to the fish tissue were equilibrated. The absence of reference tissue containing the analytes being investigated made the development of a spiked tissue material necessary. Historically the dosing of analytes to tissue was performed by spiking the analytes into aquariums containing fish and sacrificing the fish after an exposure period. The aquarium approach was not practical for this study considering the number of samples for analyses and the number of analytes to be determined. Evaluation of the analyte-tissue interaction period was required to assure the analytes are in equilibration with the matrix.

It was reasoned that the response relative to a standard (relative response) of an analyte produced by the vacuum distillation of fish tissue was respective to its sorption with the matrix. If the relative response of an analyte continues to decrease as the time of its contact with the matrix was increased, there was not yet a state of equilibration. The analyte was considered at equilibrium with the tissue when its relative response no longer decreased when the equilibration interval was increased.

Complicating the determination of analyte recovery from the tissue samples, however, was the enhanced distillation rate of water (almost double that of water without tissue) when 1 g tissue was added to 5 mL water. The tissue and water mixture boiled more vigorously than just the 5 mL water resulting in the response of some analytes to increase while others decreased. The effects due to the vacuum distillation (not degree of equilibration) were reduced by calibrating the VD/GC/MS system using standards in a fresh tissue and water mixture. The standard with tissue was prepared immediately before analyses to reduce equilibration in the standard. The minimization of analyte response as compared to the fresh standards (relative response) was then assumed to be due to the tissue effects enhanced by extending the equilibration period.

By lengthening the equilibration period until there is no subsequent drop in analyte relative response it was assumed the matrix and spike were in equilibration. Replicate analyses were performed to determine average analyte relative responses for various equilibration times. Figure 1 illustrates the relationship of analyte response to equilibration times for naphthalene-d₈. Naphthalene-d₈ was representative of compounds that required the longest time for equilibration with the tissue.

Three types of spike techniques were investigated for this study. These techniques were the water spike (the spiking technique commonly used by the analytical community to add VOCs to a sample), a sonication spike, and vacuum spike. Initially samples were vacuum distilled using 1 g aliquots of tuna and 5 mL of water. After the initial equilibration studies were conducted, the volume of water was reduced to 1 mL.

Vacuum spike. When tissue was vacuum spiked with analytes, the relative responses for the analytes decreased rapidly with increased equilibration time. The analytes were equilibrated with 1 g tissue after 2 hr as there was little additional decrease in relative response after 64 hours of equilibration (Figure 1).   Comparing the relative response of naphthalene-d₈ to the three different spike techniques for 1 g tissue, the vacuum spike was seen to be the superior technique as it had a reasonable time requirement and a clear equilibration end point. The water and sonication spike techniques would be evaluated as comparable to the vacuum spike.

The effectiveness of the vacuum spike for 10 g samples was also evaluated. This evaluation was done by conducting two separate vacuum spikes to the fish tissue. The first vacuum spike contained analytes for a 2-hr equilibration interval. After the equilibration period a vacuum spike of the containing deuterated analogs of the analytes was conducted. The second vacuum spike had equilibration periods of 125 to 211 min. It was assumed that when the relative response of the analyte and the relative response of its deuterated analog were equal then the compounds were in equilibrium with the tissue matrix. This study confirmed that most analytes were equilibrated after 2 hr with the relative response of the highest boiling analytes (>200 °C) within 50 and 80% of their labeled analogs. After 3 hr of equilibration, the higher boiling analytes relative responses were better than 90% compared with their labeled analogs. Evaluating the relationship of equilibration (measured as recovery of the analyte to the relative response of its deuterated analog) to the analytes boiling point, molecular weight, K_ow and K_oa it was seen that the boiling point has the greatest influence.

The vacuum spike technique demonstrated an equilibrium of analytes and 1 g tissue after 2 hrs. The vacuum spike of 10-g tuna was less equilibrated at 2 hr. Extending the vacuum spike to a 3-hr analyte-matrix interaction produced an effective equilibration spike technique.

Water and sonication spike. The water and sonication spike techniques could not be evaluated using the given assumption (equilibration was reached when relative responses of analytes had reached their minimum) as the relative responses of some analytes continued to drop after 64 hrs (Figure 1).  The sonication spike demonstrated a quicker drop in analyte relative response compared with the water spike equilibration time (Figure 1) but, after 20 hours the water and sonication spike techniques resulted in very similar analyte responses. The longer equilibration periods resulted in severe tissue degradation which was undoubtedly impacting the analyte recoveries.

The effectiveness of the sonication spike was then evaluated using two separate spikes (one for the labeled analytes and one for the unlabeled analytes) similar as that done to evaluate the vacuum spike technique. For this study the unlabeled analytes were first vacuum spiked for 2 hr. At the completion of the vacuum spike 1 mL water and the deuterated analogs (surrogates) were added to the tissue and the sonication equilibration performed. By extending the sonication period until the relative response of analyte to the relative response of their labeled analog were equal then the time necessary for spike-tissue equilibration using sonication could be established. An overnight equilibration period (>1000 min) was necessary to bring the differences (between unlabeled and labeled analogs) in relative responses to less than 10%. A similar comparison of the water spike to the vacuum spike was conducted and 1000 min water spike equilibration was found equivalent to the 1000 min sonication equilibration.

The vacuum spike was the most desirable technique. The vacuum spike equilibrated all analytes with tissue in less than three hours and was effective for sample sizes as large as 10 g. The use of the sonication technique was good when 1 g samples were to be analyzed (1 mL water) and the equilibration period could be more than 1000 min. The water spike was also adequate for 1 g samples if the equilibration period was extended, however, the additional water necessary to mix the sample was undesirable.

The sonication and vacuum spike techniques were used to evaluate the surrogate-correction models with the sonication spike technique used for 1 g tissue samples and the vacuum spike technique used for 10 g tissue samples.

Surrogate-Correction Model 1. One condition for applying the VD/GC/MS method with surrogate correction was that the surrogates must accurately describe matrix effects on analyte recoveries. The previously reported surrogate-matrix effects model (Model 1)^4,5 using vapor pressure (measured as boiling point) and relative volatility was evaluated for application to tissue samples. Model 1 incorporates surrogates to monitor and correct relative volatility effects (vaporization from a sample) through the relationship

R_w = e^aa_Kw ^{+ c}                     (1)

where R_w is an analyte's recovery corresponding to its a_Kw value, a and c are constants, and a_Kw is the relative volatility of the analyte.

An additional function describing the recovery of analytes with losses due to condensation is

R_B = b(bp-bp₀) + d                 (2)

where R_B is the recovery corresponding to the boiling point, b and d are constants, bp is the analyte's boiling point, and bp₀ is the boiling point where effects were shown to be negligible (80°C).

The recovery of an analyte is then calculated as R_w × R_B resulting in the equation

Recovery = e^aaK_w ^{+ c} ( b (bp-bp₀) + d)                         (3)

The vacuum distillation of a sample that contains four or more surrogates provides the information necessary to solve Eq 3. Using the experimental recoveries of the surrogates (relative responses), their boiling points, and their relative volatilities (a_K) provide solutions for the constants. The determination of the constants can be simplified by solving Eq 1 or Eq 2 separately using the recoveries of surrogates with either boiling points or relative volatilities that are equal. The recovery of any other analyte is then be predicted using its boiling point and relative volatility to solve Eq 3. In practice the surrogates are grouped to solve increments of the range of boiling points and relative volatility values representing the analytes. It was noted that by using more surrogates in a grouping than necessary to solve the equations would provide replicate predicted recoveries for analytes with their variation an estimate of the error associated with the solutions for Eq 3.

This approach worked very well when 1 g tissue samples were analyzed Table 2.  The method detection limits were lower than those previously reported for fish tissue and improved matrix-corrected recoveries.² The recoveries reported in Table 2 were a means to gauge the accuracy of the surrogate corrections. Recoveries in Table 2 and Table 4 were calculated as the experimental relative response of analytes divided by their predicted relative response, 100% recovery being complete agreement. The data presented in Table 2 was generated using analyte responses compared to aqueous standards. Using standards prepared in a tissue matrix had little impact on the results for most analytes. The basic semivolatile analytes were found to have improved determinations when their standards were prepared in a tuna matrix.

Surrogate Correction Model 2. When 10 g samples were analyzed with matrix corrections using Model 1, the recoveries (experimental relative response divided by predicted relative response) of some analytes were less desirable. Another surrogate correction model (Model 2) was developed to improve the prediction of matrix effects for the larger samples. Model 2 still incorporated the Model 1 function, R_w, to describe analyte recovery from a water phase but added a new function to describe the partitioning of the analytes between the organic phase and the vapor phase.

The partitioning between an organic phase and the vapor phase during a vacuum distillation is described in the same manner as the water-air partitioning⁴ using the relative volatility from an organic phase. The relative volatility of analytes from an organic phase made to correspond to the calculated K_oa partitioning values is defined in this work as a_Ko.  Before Model 2 could be tested, values for K_oa and the relative volatilities of the analytes from an organic phase were determined.

The fugacity capacity coefficients (Z) for air and water and their relationships to partition coefficients are described as K_ow = Z_o/Z_w, K_wa = Z_w/Z_a, and K_oa = Z_o/Z_a.⁷ The octanol-water partition coefficients are then calculated as

K_oa = K_ow K_wa                     (4)

Using the relative volatility values previously reported (a_Kw) as K_wa and the K_ow values listed in Table 1, values for K_oa were calculated and the results listed in Table 1.  The calculated K_oa were used as a basis to assign values for a_Ko.

The relative volatilities of analytes from an organic phase were experimentally determined similar to the determination of relative volatilities from a water phase.⁵ In that work successive vacuum distillations were performed on a sample. The response for an analyte in the first vacuum distillation compared with the sum of analyte responses from the successive distillations (recovery) corresponded to their relative volatilities. Using analytes with known K_wa the relationship of recovery to relative volatility was described in terms of K_wa and analytes were assigned values for their a_Kw.

The experimental a_Kovalues were extrapolated from a relationship of calculated K_oa to analyte recovery from an organic phase by vacuum distillation. The recovery of an analyte was determined by comparing the response of an analyte to its response if 100% was detected. The 100% response was determined by performing sequential analyses (between three and six analyses) of 0.2 g cod liver oil spiked with the analytes. Some analytes were completely removed from the oil phase by the series of vacuum distillations and the 100% response was simply the sum of their responses. The 100% response for the remaining analytes was calculated as the sum of responses including predictions of responses that could be obtained with additional vacuum distillations (extrapolating the trend of responses). The analyte responses from the first analyses were then divided by the 100% response to determine the recovery of analyte for the first analyses. Using calculated K_oa for chloromethane, benzene-d₆, toluene-d₈, ethylbenzene, o-xylene-d₁₀, naphthalene-d₈ and methylnaphthalene-d₁₀ to define the recovery rate-to relative volatility (organic phase) relationship, experimental relative volatilities from organic phase values for all of the analytes were extrapolated. The resultant a_Kovalues are also listed in Table 1 and these values were used in surrogate correction Model 2.

The relationship of analyte recovery respective to relative volatility (a_Ko) is the same general equation as the vapor-water phase partitioning equation used in Model 1. Incorporating the organic phase variables, the relationship is described as

R_o = e^ba_Ko^{+ d}                     (5)

where R_o is analyte recovery corresponding to its volatilization from the organic phase, b and d are constants, and a_Ko is the relative volatility of the analyte.

Model 2 does not include the effects of condensation as a separate function. The effects corresponding to a_Ko are much greater and render the condenser-boiling point corrections contained in Model 1 less important. Treating the condenser-boiling point and a_Ko effects separately becomes even less important considering the close correlation between K_oa and boiling point for organic compounds.

The determination of analyte recoveries from a two-phase sample required addressing the partitioning of analytes between the water and organic phases. Model 2 was developed with the assumptions that the analytes were at equilibrium between the phases and additional equilibration of analytes between the organic and water phases are insignificant during a vacuum distillation. The partitioning of the analytes between the phases was calculated using the analyte K_ow and, for this study, approximating the water and organic content as 95 and 5%, respectively. The following equation is descriptive of analyte recovery calculations used by Model 2

R = R_w + R_o = X_w × e^{aaKw + c} + X_o × e^{baKo + d}         (6)

where R is the recovery of an analyte from tissue and X_w and X_o is the fraction of analyte contained in the water and organic phases (X_w + X_o = 1). Solving the equation for an analyte requires solutions for the constants, a-d. In a manner similar to a previous study, these equations were solved for range of values using specific surrogates (Table 3) and their respective experimental recoveries (relative response). The solutions for R_o equations were first solved using surrogates that have elevated K_ow where losses are almost entirely due to the K_oa partitioning. Using the multiple surrogates in each range of K_oa values (four solutions), R_o is determined with a confidence interval for all analytes. With the R_o solutions determined for the four ranges (Table 3), R_w relationships are solved for the three ranges of a_Kw-values using their representative surrogate pairs. As with R_o multiple determinations of R_w provided confidence intervals for each range.

Results for the analyses of 1 g tissue samples (sonication spiked with overnight equilibration) and 10 g tuna samples (vacuum spiked with overnight equilibration) are provided in Table 4.  There was minimal difference in analytical results for Model 1 and Model 2 when 1 g tissue was analyzed and suggests the models are equivalent for 1 g samples. Model 2 was more effective than Model 1 for those analytes that had K_oa boiling point relationships dissimilar to that for the bulk of analytes. The major advantage of Model 2 is that a larger sample can be analyzed without compromising the accuracy of matrix corrections. Comparing the analyses of 10-g tissue to standards in water yields the accurate prediction of analyte recoveries (experimental relative response divided by predicted relative response). The low relative responses (less than 10%) for the neutral semivolatile analytes were observed for 10-g samples; however, recoveries of 90 ± 16 % illustrate the ability of Model 2 to describe and compensate for the matrix effects. The analyses of 10 g samples provide MDLs that are an order in magnitude lower than previously reported.

Spectral interferences for the analytes studied were infrequent. The coelution of methanol and water caused periodic chromatography and integration problems for several early eluting compounds (ethyl ether, acetone, 1,1-dichloroethene, iodomethane, allyl chloride and methylene chloride), but the surrogates, ethyl ether-d₁₀ and methylene chloride-d₂ were good monitors for this effect.

Several analytes used in this study were found unacceptable as analytes in tissue. These compounds apparently reacted with the tissue matrix and formed adducts that were not detected. Iodomethane and bromomethane disappeared at rates greater than 99% and 90 +/- 2% per day respectively for a 10-g tissue sample. Halomethanes have been reported as being active alkylating agents with hemoglobin and explains their disappearance in fish tissue.⁸ The allyl halides also appeared to react with the tissue (presumably due to nucleophilic substitution and as these compounds are strong alkylating agents). All the allyl halides in this study disappeared quickly at the following rates (percent per day): allyl chloride, 24.1 ± 14.5; trans-1,3-dichloropropene, 72.6 ± 5.6; cis-1,3-dichloropropene, 56.7 ± 4.7; cis-1,4-dichloro-2-butene, 97.3 ± 1.3; and trans-1,4-dichloro-2-butene, 77.9 ± 4.3.

Acrylonitrile was found to degrade at a rate of 47 +/- 25% per day for a 10-g tissue sample. Acrylonitrile has been found to bind to proteins irreversibly, also a likely source of degradation in fish tissue.⁹

The concentration of pentachloroethane used in this study was near its MDL and its results were not included in Tables 2 or 4. 1,1,2,2-Tetrachloroethane coeluted with compounds codistilled from the tissue and interfered with determining its response. This analyte was not used in the summaries presented in Tables 2 and 4. Both analytes are listed in Supporting Information.

The basic semivolatiles were generally the poorest performing group of analytes in this study. Surprisingly the performance data for this group was best for 10 g samples (using standards in water) and using Model 2 for surrogate correction of matrix effects. The performance data for this group would most likely be improved by using basic semivolatile surrogates (there were none used in this study). The soluble volatiles were the poorest performing analyte group for 10 g samples. The performance of this group would most likely be improved by narrowing the range of a_Kw values that a given surrogate-pair represents (adding surrogate-pairs and new ranges for corrections). These two groups of analytes have low BCFs (a lower priority for this study) and additional surrogates were not investigated.

The development of fish tissue samples in which analytes have demonstrated equilibration provides a medium to develop and evaluate methods for fish analyses. The analysis of these samples demonstrates VD/GC/MS using surrogate-based matrix corrections can be applied to difficult matrices with an accuracy and sensitivity comparable to analysis of reagent water. The measurement and correction of the most extreme matrix effects using calibration standards in water verify the correctness of the models. The method is shown to be applicable to both simple and difficult samples without the need for special calibration requirements.

The analyst has two surrogate-correction models to compensate for matrix effects on analyte response. Model 1 is appropriate for most analytes when analyzing 1-g samples. Model 2 is recommended for larger samples where lower MDLs are required. The calibration of the VD/GC/MS system is adequately performed with standards in water.

ACKNOWLEDGMENT

The EPA, through its Office of Research and Development (ORD), funded and performed the research described here. It has been subjected to the Agency's peer review and has been approved as an EPA publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. The U.S. Government has the right to retain a non-exclusive, royalty-free license in and to any copyright covering this article.

SUPPORTING INFORMATION AVAILABLE

The large amount of data used in the development of this work could not be presented in its entirety due to space restrictions but additional data are available. The data (by analyte) used to prepare Tables 2 and 4 are available as two additional tables. Figure 1 represents the equilibrium profile for only one compound. The corresponding data for all of the analytes are available as three additional tables. The labeled-unlabeled analog data used to evaluate the effectiveness of the sonication spike of 1 g tissue and the vacuum spike of the 10 g tissue samples are available as two additional tables. 

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