WAS LINDANE ISOLATED AND RECOVERED FROM A BIOLOGICAL MATRIX USING RP-SPE? IF SO, HOW?

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eluted off of the sorbent with two 500-µL aliquots of pesticides-residue-grade isooctane. The iso-octane eluent was then passed through a second SPE cartridge. This

second cartridge was packed with approximately 0.5 g of anhydrous sodium sulfate.

The volume of eluent that now contained the recovered analyte was adjusted to a final

volume of 1.0 mL using a volumetric flask. Three microliters of this eluent was injected

via autosampler into an Autosystem Gas Chromatograph (PerkinElmer) incorporating

an electron-capture detector. A 30 m × 0.32 mm capillary GC column containing DB-5

(J&W Scientific) was used to separate the organics in the eluent. This instrument is

abbreviated C-GC-ECD to distinguish it from other gas chromatographs in our laboratory. The column was temperature programmed following injection from 200 to

270°C at a rate of 10°C/min. The C-GC-ECD is connected to a 600 Link (PE-Nelson)

interface module. This interface, in turn, is connected to a 386 personal computer. This

PC uses Turbochrom® (PE-Nelson) software for data acquisition, processing, and

control. A method specific for lindane was written, and one peak was identified within

a 3% relative time interval (retention time window). A retention time, t[R], of 1.8 min

was consistently reproduced using autosampler injection.



Calibration

A series of calibration or working standards were prepared from the methanolic stock

solution containing lindane. This stock solution was prepared by carefully weighing

out pure solid lindane on an analytical balance. These standards were injected into the

C-GC-ECD from lowest to highest lindane concentration. The peak at 1.8 min was

identified as a reference peak in the Turbochrom software, and a narrow t[R] window

was defined around the t[R] of the apex of the peak. A calibration curve was constructed

using a least squares regression algorithm in the software. A correlation coefficient of

0.9990 was obtained.



Sample Analysis

The sequence within which the samples were run after calibration was created by using

the Sequence File Editor within Turbochrom. Samples are injected via autosampler

according to the instructions in the Sequence File. After the sequence is completed, a

Summary File is created and the analytical results are reported within this summary

format. In the Summary File, for each sample analyzed, the following is given:

(a) The sample number

(b) The concentration of lindane in ppb for 1.0 mL of eluent, interpolated from the

external standard mode of instrument calibration

(c) The retention time for lindane, t[R], in minutes

(d) The integrated peak area, in microvolts-seconds

The reported concentration is that for each eluent and should be multiplied by the

eluent volume (in this case, 1.0 mL) to obtain the number of nanograms of lindane

found. The number of nanograms found divided by the volume of the aqueous sample

used in RP-SPE gives the reported concentration in ppb for lindane. The reported

concentration should be divided by the volume of the sample to obtain the reported

concentration in ppb for lindane. The reported concentration should also be divided

by the percent recovery, expressed as a decimal, in order to find the true and final

concentration of lindane in the original sample.

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Sample Preparation Techniques



247



Method Evaluation

Lindane is efficiently recovered from aqueous matrices with percent recoveries between

75 and 100% using RP-SPE. The biological sample closely approximates an aqueous

matrix.



70. DOES IT MATTER WHICH ELUTION SOLVENT

IS USED IN RP-SPE?

Answers to this can be given as yes and no and a qualifying statement that it might

depend on the polarity of the retained analyte. With a series of analytes that have

similar octanol–water partition coefficients, differences in eluting solvent polarity

may not significantly influence analyte recovery. If breakthrough studies are not

performed, as discussed earlier, one never knows whether low percent recoveries

are due to poor sorption or to inefficient elution or solvent removal using the RP-SPE

technique. This author recently had the opportunity to quantitatively determine trace

concentrations of 2,4,6-trichlorobiphenyl (246TCBP) and 4-hydroxy-2,4,6-trichlorobiphenyl (4HO246TCBP) in biological matrices. Molecules from these two organic

compounds of environmental interest differ in that a hydroxyl group is substituted

for a hydrogen on the phenyl ring para to the second phenyl. These structures are

as follows:

Cl



Cl



Cl

2, 4, 6-trichloro-bipheny1

Cl



Cl



O

H

Cl



4-hydroxy-2, 4, 6-trichloro-bipheny1



We were quite surprised to measure a zero percent recovery when we attempted

to apply our method for OCs to isolate and recover 4HO246TCBP from spiked

water. Our recoveries of 246TCBP using our OC method were very high. Replacing

iso-octane with methanol as the eluting solvent resulted in a significant increase in

percent recoveries for 4HO246TCBP. This surprising finding led us to conduct a

series of systematic RP-SPE recovery studies. An aqueous spiked sample was prepared that contained both PCB congeners, and two elution schemes were used. In

Scheme A, the retained analytes were eluted with methanol followed by a separate,

second elution with iso-octane. Two receivers were used, and their final volumes

were both adjusted to a 1.0-mL total eluent volume. In Scheme B, the retained

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Trace Environmental Quantitative Analysis, Second Edition



TABLE 3.13

Comparison of Two Sample Preparation Schemes to

Isolate and Recover 2,4,6-Trichlorobiphenyl and 4Hydroxy-2,4,6-Trichlorobiphenyl Using RP-SPE

246TCBP

Scheme A

Elute with

Elute with

Scheme B

Elute with

Elute with



4HO246TCBP



MeOH, mean of 4 SPEs

iso-octane, mean of 4 SPEs



137,026

40,413



218,526

58,259



iso-octane, mean of 4 SPEs

MeOH, mean of 4 SPEs



210,234

24,825



0

194,002



analytes were eluted with iso-octane followed by methanol. The integrated peak

areas (in units of µV-sec) for the 1-mL eluents are represented in Table 3.13. It is

a good assumption that the response factors for both congeners are near one another

because the substitution of a hydroxyl for hydrogen in the phenyl ring contributes

little or nothing to the ECD response. Eluting first with two 500-µL aliquots of methanol

recovers more 4HO246TCBP than 246TCBP, whereas the second elution with isooctane recovers nearly equal amounts of both congeners. Thus, Scheme A reflects

incomplete recovery of the congeners with two 500-µL aliquots of methanol. Eluting

first with two 500-µL aliquots of iso-octane recovered even more 246TCBP than MeOH,

yet recovered no 4HO246TCBP. This observation was consistent with our preliminary

finding, discussed earlier. Upon eluting with MeOH via Scheme B, a relatively small

amount of 246TCBP was recovered, whereas a relatively large amount of

4HO246TCBP was recovered. The percent recoveries for 4GO246TCBP were similar

between Schemes A and B when MeOH was used to elute off the retained analyte.

Another illustration from the author’s experience is the differences in percent

recovery when hexane is used vs. methyl-tert-butyl ether (MTBE) to elute off of a

C18 sorbent. Two different aqueous matrices were spiked with the OPs diazinon and

malathion. In one series of experiments, two 500-µL aliquots of hexane were used

to elute off of the cartridge, followed by two 500-µL aliquots of MTBE eluted off

of the same cartridge. The same experiment was performed on a second aqueous

matrix that was more acidic by virtue of adding potassium dihydrogen phosphate

(KH2PO4). The results are shown in Table 3.14. It is clearly evident that the more

polar MTBE more effectively removes both OPs from the hydrophobic sorbent than

the less polar hexane. This fact has perhaps more to do with the polarity of OPs and

the nature of the sorbent–analyte–solvent triangle, as discussed earlier in this chapter.



71. HOW CAN PCBS BE ISOLATED AND RECOVERED

FROM SERUM, PLASMA, OR ORGAN FOR TRACE

ENVIRO-HEALTH QA?

Scheme 3.1 (shown earlier) is a flowchart that summarizes this author’s attempt to

use RP-SPE as part of a combination of sample prep techniques that isolate and

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249



TABLE 3.14

Percent Recoveries for Diazinon and Malathion Using

Hexane and MTBE as Elution Solvents in RP-SPE

Diazinon

Matrix: 10% NaCl (DDl)

1. Elute with hexane, mean of 3 SPEs

%RSD

2. Elute with MTBE, 1 SPE

Matrix: 10% NaCl, 0.2 M

1. Elute with hexane, mean of 3 SPEs

%RSD

2. Elute with MTBE, 1 SPE



Malathion



20

18.3



0

—



20

14

112



0

—

98



recover AR 1248 with nearly 100% recoveries.101 PCBs are easily released from a

serum, plasma, or tissue homogenate via probe sonification (PC), coagulation using

acetonitrile, or other water-miscible organic solvent or salt. The supernatant aqueous

phase is easily separated from the coagulated protein via mini-centrifugation.

RP-SPE serves in this case, similar to the situation when an environmental water

sample is passed through, as an on–off or digital extraction step. The dilute of the

aqueous supernatant serves to decrease the analyte–matrix interaction, as discussed

earlier, which in turn strengthens the analyte–sorbent interaction. In this case, the

combination of PS-RP-SPE with C-GC-ECD as the determinative technique eliminated the need for further cleanup. Such might not be the case if C-GC-MS or

another universal determinative technique is used. Cleanup techniques might then

need to be considered. Listed below are significant outcomes from the author’s

method development study:101

Sample



Recovery as Total PCB (%)



Spiked

Spiked

Spiked

Spiked

Spiked



102

102

115

88.2

77.7



acetonitrile 1

acetonitrile 2

rat liver 1

rat liver 2

rat liver 3



Janák and coworkers102 have discussed a similar approach to the isolation and

recovery of PCBs from whole blood. Details of their approach are outlined via a

flowchart in Scheme 3.8. Referring to Scheme 3.8A, formic acid dissolved in isopropyl alcohol (IPA) was used to coagulate protein from 5 g of whole blood prior

to bath sonication. After passing the supernatant through a conditioned RP-SPE

cartridge, the sorbent is washed with 5% IPA dissolved in water, followed by 10%

methanol in water. The sorbent is then washed again with a series of solutions as

indicated and dried. Referring to Scheme 3.8B, the sorbent is eluted with methylene

chloride (dichloromethane), then evaporated down to 10 µL, and finally reconstituted

with enough heptane to yield ~100 µL. This heptane eluent is transferred to a Pasteur

© 2006 by Taylor & Francis Group, LLC