A new method for purifying fat containing extracts in the determination of polybrominated diphenyl ethers

AnChem1904013Shelepchikov fm ISSN 1061 9348, Journal of Analytical Chemistry, 2019, Vol 74, No 6, pp 574–583 © Pleiades Publishing, Ltd , 2019 Russian Text © The Author(s), 2019, published in Zhurnal.

A New Method for Purifying Fat-Containing Extracts

in the Determination of Polybrominated Diphenyl Ethers

A A Shelepchikova, b, *, V V Ovcharenkoa, A I Kozhushkevicha, E S Brodskiib,

A A Komarova, K A Turbabinaa, and A M Kalantaenkoa

a The Russian State Center for Animal Feed and Drug Standartization and Quality, Moscow, 123022 Russia

b Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Moscow, 119791 Russia

*e-mail: dioxin@mail.ru

Received November 13, 2017; revised July 1, 2018; accepted July 1, 2018

Abstract—We developed a sample preparation method for the determination of polybrominated diphenyl

ethers (PBDEs) with from one to ten bromine atoms in samples of feed and food products containing approx-imately 0.5 g of animal fat or vegetable oil The method involves gas chromatography with high-resolution mass spectrometry or tandem mass spectrometry A possibility of using various reagents for the purification

of extracts by chemical reactions and fractionation is studied The physicochemical properties of PBDEs and polychlorinated biphenyls (PCBs) have significant differences, and to determine the full range of PBDEs, it

is necessary to use other methods of sample preparation than in the case of PCBs The conditions selected for the purification of extracts in a column filled with potassium silicate, Florisil, and silica impregnated with sulfuric acid and for their fractionation using activated neutral alumina ensure the PBDE recoveries of at least 75% Purification of the extracts can be carried out without the use of chlorinated organic solvents Applied aspects of instrumental analysis and measurement quality assurance are also described.

Keywords: polybrominated diphenyl ethers, sample preparation, fractionation, food and feed, biological

sam-ples, organic pollutants

DOI: 10.1134/S1061934819040130

Polybrominated biphenyl ethers are products of

target industrial synthesis and are used to reduce the

f lammability of polymeric materials The production

of PBDEs began in the 1970s in Germany There are

three main industrial products: penta-, octa-, and

decabromodiphenyl ether (DeBDE) The last product

is mainly used in the electronics industry, accounting

for approximately 82% of world production; the other

two compounds are congener mixtures used in the

plastics industry and in the furniture industry [1, 2]

There are 209 of PBDE congeners in total; they

contain from one to ten bromine atoms Because of

cumbersome names of the systematic nomenclature,

the PBDE names use arithmetic numbers that

coin-cide with the IUPAC numbers for polychlorinated

biphenyls with substituents in the same positions of

aromatic rings [3] For example, BDE-99 corresponds

to 2,2',3',4',5-PeBDE; to denote isomer groups in

bro-mination degrees, conventional prefixes derived from

the roots of Greek and Latin numerals (mono-, di-,

tri-, tetra-, etc.) and abbreviations (MoBDE, DiBDE,

TrBDE, TBDE, etc.) are used

Active studies of environmental pollution and

bio-logical samples with PBDEs began approximately 20

years ago [4] The results of these studies were the

rea-son for banning or restricting the use of PBDEs in the United States and the European Union In 2009, tech-nical mixtures of penta- and octabromodiphenyl ethers were included in the expanded list of the Stock-holm Convention on Persistent Organic Pollutants; DeBDE is a candidate for inclusion in this list Con-ventional approaches to the determination of these substances have not been formed By their structure PBDEs are similar to PCBs, and it can be assumed that the methods for their isolation are similar In practice, this is true only for medium-brominated compounds, which are the most common substances for determination [5] Highly brominated congeners, including DeBDE, are determined less often because

of difficulties in chromatographic separation of these compounds Mono- and dibromodiphenyl ethers are determined even less often, and the authors of [6–8], recognizing the possibility of their determination, point out the problem of low recoveries or do not give them at all for MoBDE

This article is devoted to the development of a method of the purification of fat-containing extracts, which enables the determination of PBDEs with any degree of bromination and monitoring the level of contamination of feed and food Other applied aspects

ARTICLES

of analysis and their relationship with the sample

preparation procedure are also described

PECULIARITIES OF DETERMINATION

OF POLYBROMINATED DIPHENYL ETHERS

Like most methods of organic trace analysis, the

determination of PBDEs in various samples consists

of three main stages: extraction, purification

(separa-tion of the target compounds from other extracted

components of the matrix), and instrumental analysis

Extraction from samples of animal lipophilic organic

pollutants including PBDEs actually comes down to

the extraction of fat, which is a relatively simple task

It is more challenging to extract PBDEs from samples

of plant origin, to which the analytes pass from the

atmosphere or from the soil In both cases, extraction

techniques that have proven effective for PCBs or

polychlorinated dibenzo-p-dioxins and dibenzofurans

(PCDD/PCDF) can be used; there are no

prerequi-sites to assume that the extraction method, useful for

PCBs and even more so for PCDD/F, would be

unsuitable for PBDEs In this regard, we do not

con-sider the stage of extraction in this paper

The size of a sample and the corresponding

proce-dure for the purification of extracts depend on the

sen-sitivity requirements of the method of quantitative

determination and the available measurement

equip-ment The levels of PBDEs in samples can be

conven-tionally described as significantly lower than the levels

of PCBs but higher than those of PCDD/PCDF The

primary method for determining PBDEs is gas

chro-matography–high-resolution mass spectrometry

(GC–HR-MS), a method used to quantify

PCDD/PCDF when the highest sensitivity

require-ments are imposed

Because of the high sensitivity and selectivity of

GC–HR-MS, small samples with minimal

purifica-tion can be analyzed, for example, by passing the

extract through a Pasteur pipette filled with silica

impregnated with sulfuric acid and/or filtering the

sample through activated silica or Florisil Such an

approach not only saves solvents and adsorbents but

also helps to decrease contamination of the blank

sample because PBDEs are present in almost all

sol-vents and adsorbents In our case, this technique is not

applicable, since it is necessary to be able to work with

samples containing a sufficiently large amount of fat

Because of the structural similarity of PBDEs and

PCBs, it is usually proposed to use techniques that

have previously been tested for PCBs for purification

of extracts The main method can be considered the

destruction and adsorption of labile matrix

compo-nents on a multilayer column consisting of layers of

silica impregnated with sulfuric acid and potassium

hydroxide or silicate, separated by anhydrous sodium

sulfate, and fractionation on alumina, when the

ana-lytes are eluted with a mixture containing several

per-cents of dichloromethane (DCM) in hexane A similar purification algorithm is implemented in the most well-known automatic sample preparation system from FMS (Waltham, United States) However, when analyzing fish meal samples using the FMS Total-Prep system according to the procedure proposed by the manufacturer, we obtained recovery rates of 44– 77% for medium-brominated BDEs; MoBDE was absent in the extract, and the recovery of DiBDE did not exceed 15% The contamination level of the blank BDE-47 sample was approximately 20 pg when using specialized FMS disposable columns for PBDEs Other users of FMS systems also encountered the problem of losing low-brominated congeners [9, 10]

A radical increase in the volume of solvents and the use of pure dichloromethane instead of its mixture with hexane does not allow the recovery to reach even 10% for MoBDE [11] The procedure recommended

by the Ministry of the Environment of the Canadian Province of Ontario, using the FMS automatic sample preparation system, ensures the determination of PBDEs containing at least three bromine atoms [12] The developers of Method 1614, which is an official method for determining PBDEs of the US Environ-mental Protection Agency, also probably faced the problem of extracting MoBDE and DiBDE, as indi-cated by the absence of criteria for assessing recovery rates for these compounds [13] However, they did not use the automatic sample preparation system but pro-posed a procedure similar to Method 1668 for deter-mining PCBs [14]

A separate problem in the determination of PBDEs

is their chromatographic separation: in addition to the absence of columns capable of separating all existing isomers [15, 16], these substances have low volatility with insufficient thermal stability Polybrominated diphenyl ethers containing up to 5–6 bromine atoms can be determined by GC–MS using DB-5ms column (5% of 1,4-bis(dimethylsiloxy)phenylenomethylpoly-siloxane) or HT-8 columns (8% of phenylpolycarbon-ate siloxane), 25–30 m in length with a stationary phase layer thickness of 0.22–0.25 μm, typical for determining PCDD/PCDF or PCBs In the case of heavier congeners, a sharp decrease in sensitivity is observed up to the complete disappearance of chro-matographic peaks There is information [15] on the determination of BDE-209 using long columns with different stationary phases; however, the amount of substance injected into the chromatograph must be taken into account In our experience, the problem of chromatography of BDE-209 resembles the situation with DDT, when some constant amount of substance

is subject to thermal decomposition; that is, the higher amount of the substance introduced, the smaller the relative loss In order to determine subnanogram quantities of highly brominated PBDEs reliably, it is advisable to use a special J&W DB-5ht chromato-graphic column 10–15 m in length with a thinner layer (0.1 μm) of inert stationary phase (95% of

methylsi-loxane, 4% of phenylsimethylsi-loxane, and 1% vinylsiloxane).

Using such a column, PBDEs with any degrees of

bro-mination can be detected, but the quality of the

sepa-ration of the isomers could be rather low, and a

sub-stantial distortion of the peaks in the initial part of the

chromatogram could occur However, the insufficient

purification of the extracts usually does not affect the

highly brominated compounds To obtain reliable

quantitative results, at least two different

chromato-graphic columns should be used

EXPERIMENTAL The published data and our experience show that

the loss of low-brominated diphenyl ethers occurs

during fractionation on activated alumina and they

may be absent in simplified methods, for example,

when removing the bulk of fat by freezing and further

purification in a multilayer column [7] or using gel

chromatography [17] Unfortunately, these and other

options for purification of extracts without

fraction-ation cannot be considered as universal methods for

the routine determination of traces of PBDEs in

fat-containing matrices

There are data [18] on low, but not zero recoveries

of mono- and dibrominated diphenyl esters, obtained

using alumina cartridges for the purification of soil

extracts or adding it to the cartridge for accelerated

solvent extraction; however, no information about the

brand of cartridges or alumina is given There is also

no data on cartridges used in the procedure for

deter-mining PBDEs of any degree of bromination in milk

[19]

The inability to elute MoBDE from activated basic

alumina with dichloromethane and toluene

quantita-tively suggested that debromination or another

chem-ical transformation of the substances takes place; in

other words, the basic principle of quantitative

analy-sis method, i.e., the absence of chemical reactions

(except target derivatization) between the substances

to be determined and the reagents used, is violated We

decided to create a new procedure for determining the

full range of PBDEs rather than to adapt the available

procedures, for which it was necessary to study the

possibility of using different adsorbents and

tech-niques

Test samples The effectiveness of the developed

procedure was tested using three types of samples:

pork fat, fish oil, and sunf lower oil They are

represen-tatives of the three main fat-containing matrices:

ani-mal, fish, and vegetable There are no values of

maxi-mum permissible concentrations or other standards

for the concentration of PBDEs in feed and food

products; the European Union directive on

monitor-ing these compounds in food products specifies a limit

of determination of no less than 10 pg/g of wet weight

[20] Considering that the same instruments are used

to determine PBDEs and PCDD/PCDF, and the

weighed portions of the latter contain no more than 3–5 g of fat in their routine determination, we can assume that 0.5 g of fat should be sufficient to estimate the concentration of PBDEs

Equipment At the preliminary stage of research, a

Thermo TSQ8000 Evo triple quadrupole was used in the MS/MS mode with a Trace 1310GC gas chro-matograph equipped with a Thermo TR-5MS column

30 m in length, 0.25 mm in diameter, and the thick-ness of the stationary phase layer of 0.25 μm A sample

of 1.5 μL in volume was injected in the splitless mode

at the injector temperature of 290°C; purging of the injector was 1.5 min after the injection The tempera-ture program of the chromatographic separation was

as follows: the initial temperature of the thermostat was 140°C; holding at this temperature for 2 min; heating to 220°C at a rate of 10 deg/min; then heating

to 245°C at a rate of 5 deg/min and to 290°C at a rate

of 10 deg/min; holding at this temperature until the end of the elution Under these conditions, ethers from MoBDE to HxBDE and sometimes HpBDE can

be determined The use of MS/MS techniques in some cases gives mass-chromatograms that are more convenient for interpretation, especially, in the case of DiBDE, the exact masses and retention times of which are close to PeCB; however, the error in determining the recoveries of isotope-labeled reference com-pounds is higher, which is described in more detail below

The remaining PBDEs were detected, and a con-firmatory determination of low-brominated conge-ners was performed using a Waters AutoSpec Premier high-performance chromatography–mass spectrome-ter with J&W DB-5ht column (length 10 m, inspectrome-ternal diameter 0.25 mm, and thickness of the stationary phase layer 0.1 μm), SGE BPX-5 column (length

25 m, internal diameter 0.22 mm, and thickness of the stationary phase layer 0.25 μm), and SGE HT-8 col-umn (length 25 m, internal diameter 0.25 mm, and thickness of the stationary phase layer 0.25 μm), con-nected to the mass spectrometer via a 2.5-m capillary with an internal diameter of 0.15 μm The temperature conditions are given in Table 1

Two characteristic isotope ions were detected for each isotope-labeled and native PBDE, the isotopic ratio was checked for correctness, and the average value of the ionic current of the isotopic cluster was calculated, which was then used for quantitative calcu-lations and for determining the recovery

Extraction was carried out using a Dionex ASE 200 and a Thermo ASE 350 accelerated solvent extractors with 33- and 100-mL cells

For chemical purification and fractionation of samples, glass columns with a length of 200 mm and

an inner diameter of 14 mm and columns with a length

of 150 mm and an inner diameter of 10 mm were used, having a narrowing on one side and a 14/23 ground

glass connector on the other side to connect to the

tank

Solvents and materials The following adsorbents

were used: basic alumina with Brockmann I activity

(Sigma-Aldrich, 199443); neutral alumina with

Brockmann I activity (Sigma-Aldrich, 199974);

neu-tral alumina, type WN-6, with Super I activity

(Sigma-Aldrich, A1522-500); Florisil (0.150–

0.250 mm, Merck, 1.12994.1001); Florisil PR (Merck,

20280); silica gel 60 (0.063–0.100 mm, Merck,

1.07734.9025), and high-purity silica (70–230 mesh,

Merck, 7754) The method of preparation of the listed

materials differed in different experiments and is

dis-cussed below; activated adsorbents were cooled to

80°C, and transferred to an airtight container, where

they were stored until use

Sodium sulfate (Acros Organics, 196640050) was

calcined for 16 h at 550°C, cooled to 80°C, and

trans-ferred to an airtight container, where it was stored until

use Potassium silicate was synthesized by adding

sil-ica to an equimolar solution of potassium hydroxide in

methanol under constant stirring; the reaction

mix-ture was left for 1 day in a desiccator Then, excess

methanol was decanted, and the product was dried

and kept for 16 h at 250°C Silica impregnated with

sulfuric acid was prepared by mixing the activated

sil-ica with conc H2SO4 to form a homogeneous mass

During the fractionation of samples, the column with

the adsorbent was conditioned with 15–20 mL of

hex-ane before injecting the sample

A mixture of isotope-labeled PBDE standard

refer-ence compounds (MBDE-MXG and PBDE-ISS-G)

and a mixture of native congeners (BRF-PAR) were

purchased from Wellington Laboratories

Solvents from various suppliers were tested for the

absence of interfering components The problem of

contamination in the blank sample is discussed below

Recoveries and quality assessment criteria for

puri-fication When using isotope-labeled internal

stan-dards, especially in the version of the isotopic dilution method, the recoveries are often not essential, and the error in their determination can be very high For example, in the determination of PCDD/PCDF, the recoveries range from 16 to 279% according to the EPA procedure [21]; a range of 60–120% is consid-ered acceptable in the European Union for the quan-titative determination of PCDD/PCDF [22] For PBDEs containing from three to nine bromine atoms, according to the EPA Method 1614, the recovery of PBDEs should be in the range from 25 to 150%, and for DeBDE, they can vary from 20 to 200% [13] Cur-rently, several mixtures of isotope-labeled internal PBDE standards are available, containing at least one isomer of each bromination degree To estimate recov-ery rates, it is proposed to use mixtures of no more than three PBDEs containing four, six, and nine bro-mine atoms (in our case, congeners 79, 138, and 206), which, because of the significant difference in masses

of characteristic ions and thermal decomposition of PBDEs inevitably increases the error of determination

of the recovery In our work with MS/MS detection, the sensitivity coefficient of 3 relative to

BDE-79 differed by 1.5–2 times on different days This effect is less pronounced for a magnetic sector instru-ment, but it is still necessary to repeat the injection of the calibration mixture regularly The distortion of the results of determination of the recovery may also occur because of the overlapping of the signals of matrix components on those of the detected substances, causing a local loss of sensitivity of the mass spectrom-eter This effect manifests itself as “subsidence” of the recoveries or their sharp increase in overlapping with the peak of the compound being determined In the isotopic dilution method, this does not lead to a dis-tortion of the quantitative results (assuming no impo-sition on the recorded ions occurs), but with the gen-eral implementation of the internal standard method, the results of the quantitative analysis may be distorted several times

Table 1 Conditions of chromatographic separation

Initial temperature, holding

time

170 ° С, 1.5 min 160 ° С, 2 min 135 ° С, 2 min

Heating, rate To 240 ° С, 20 deg/min

to 270 ° С, 15 deg/min

to 295 ° С, 10 deg/min

To 220 ° С, 8 deg/min

to 295 ° С, 6 deg/min

To 170 ° С, 15 deg/min

to 270 ° С, 3 deg/min

to 295 ° С, 5 deg/min Holding time at final

tem-perature

Sample injection mode 1 μL, splitless, 1.5 min 1 μL, splitless, 1.9 min 1 μL, splitless, 1 min

Carrier gas (helium) f low rate 1.3 mL/min, constant f low 0.8 mL/min, constant f low 0.8 mL/min, constant f low

Another source of error in determining the

recover-ies is caused by thermal destruction or other losses

during chromatography This problem is most

pro-nounced for DeBDE; in some cases, the residual

components of the matrix lower this effect, because of

which the calculated values of the recoveries can

sys-tematically exceed 100% The effect of this factor can

be estimated, and correction factors can be introduced

by comparing the change in the magnitude of the

ana-lytical signal in a series of isotope-labeled standards

used to calculate extraction rates for pure mixtures and

samples under study

In addition to the loss of PBDEs during

purifica-tion, the possibility of light-induced decomposition of

high-brominated congeners is mentioned [11] It is

also evident that MoBDE has rather high volatility,

and special attention should be paid to the

preconcen-tration of samples and their storage

Because of the high uncertainty in estimating

recovery rates in pilot experiments, we only present a

semiquantitative estimate below

Any routine analysis procedure is almost always a

compromise between purification quality, cost, and

recovery rate; the better these parameters are

bal-anced, the more effective the procedure can be

con-sidered High recovery rates alone are not an essential

requirement for routine analysis A more critical

crite-rion is their stability when working with different

matrices If one does not consider the extreme case

when the residual amount of matrix components in

the final extract is such that it is impossible to obtain

mass chromatograms, the quality of purification is a

somewhat subjective parameter In addition to the

purely visual characteristic, i.e., the absence of

stain-ing or turbidity durstain-ing the preconcentration of the

purified extract to ~10 μL, we used the following

cri-teria for assessing the quality of purification:

—no distortion of chromatographic peaks

com-pared with pure standards;

—the absence of sharp degradation of the

chro-matographic column (constancy of retention times);

—no over-peak or “humps” in the total ion current

mass chromatograms

RESULTS AND DISCUSSION

Chemical destruction of impurities We estimated

the stability of the existing isotope-labeled PBDEs by

passing them through silica impregnated with sulfuric

acid or potassium silicate at room temperature and at

85°C in a Dionex ASE 200 accelerated solvent

extraction unit The mixtures were used that were

made of silica activated at a temperature from 130 to

180°C, with the concentration of sulfuric acid from 30

to 44% The results did not show significant losses in

using potassium silicate In the case of silica

impreg-nated with sulfuric acid, PBDEs with a bromination

degree of three or higher can be considered stable For

low-brominated congeners, ambiguous results were obtained, indicating destruction at least in freshly pre-pared, highly active mixtures

The current trend in analytical practice is the use of units for accelerated solvent extraction (ASE) not only for the extraction of various samples but also for the purification of extracts or for combining both stages [5, 23] The increased temperature during ASE increases the rate of chemical destruction of the matrix; however, ASE itself and the method of extraction practically exclude the possibility of effec-tive adsorption purification A controversial point is also the efficiency of extraction with aliphatic solvents

In our case, in analyzing fish meal with different vari-ants of filling the extraction cell with silica impreg-nated with sulfuric acid, potassium silicate, and Flori-sil, the recoveries varied from 25 to ~100% with low purification quality This result could be predicted, since, based on the experience of determining PCBs,

it can be argued that the vast majority of biological samples require at least a two-stage purification com-bining chemical destruction of the matrix and frac-tionation

Fractionation For the purification of PCB and

PCDD/PCDF by fractionation, it is often recom-mended to use a basic form of aluminum oxide We conducted an experiment with a column containing

4 g of adsorbent (activated at 600°C for 16 h), with successive elution with 20 mL of hexane, 20 mL of a mixture of hexane–DCM (19 : 1, vol), and 50 mL of a mixture of hexane–DCM (2 : 3, vol) Under these conditions, PCBs enter the second fraction, except for the coplanar congeners, which, together with PCDD/PCDF, are eluted into the third fraction Mono- and dibrominated diphenyl ethers were lost; the remaining PBDEs were partitioned between the last two fractions, which shows significant differences

in the physicochemical properties of PCBs and PBDEs

Along with the basic form of aluminum oxide, the EPA methods provide for the possibility of using the acid form for determining PCB, PBDE, and PCDD/PCDF, but the developers of the methods indicate that it has less activity and offers smaller puri-fication efficiency We did not find any examples of applied use of this adsorbent; however, we checked the possibility of its use In the experiment with the acidic form of aluminum oxide (4 g, activated at 130°C for

16 h) with successive elution with 20 mL of hexane,

20 mL of a mixture of hexane–DCM (19 : 1, vol),

20 mL of a mixture of hexane–DCM (3 : 1, vol), and

20 mL of a mixture of hexane–DCM (2 : 3, vol), PBDEs were partitioned between the second and third fractions without obvious loss, that is, this adsorbent can be considered as an option for additional purifica-tion of samples

Significant differences in the properties of PBDEs and PCBs were also observed when using Florisil PR

This adsorbent is used in the determination of PCBs

and various pesticides in cases where the use of

alumi-num oxide is impossible or not effective enough [24,

25] On a column with 2 g of Florisil PR (activated at

180°C for 16 h) with the same elution sequence,

PCDEs were distributed among all fractions, while

PCBs was quantitatively eluted with hexane or a

mix-ture with a small amount of DCM in hexane

Although there is no reason to believe that a loss of

analyte substances occurs, the use of Florisil PR seems

to be unreasonable

We tested conventional Florisil (activated at 180°C

for 16 h and at 675°C for 24 h) In both cases, the

col-umns were eluted successively with 30 mL of hexane,

25 mL of a mixture of hexane–DCM (3 : 1, vol), and

40 mL of DCM In the first system, the reference

sub-stances were partitioned between the first two

fac-tions; in the second system, the main part was in the

hexane fraction, and only trace amounts were present

in the DCM fraction Thus, this adsorbent is not

suit-able for the adsorption of PBDEs from solutions but

can be used to remove other components of the

matrix

The last tested adsorbent was neutral alumina, the

use of which is not recommended by the EPA methods

for PCBs or PBDEs, but it is effective in determining

polycyclic aromatic hydrocarbons In the first

experi-ments (4 g, activated at 400°C for 16 h), there was no

leakage of PBDEs during washing with hexane, and all

reference substances were quantitatively eluted with

20 mL of a hexane–DCM mixture (4 : 1, vol);

how-ever, we later observed a loss of mono- and

dibromi-nated diphenyl ethers When the activation

tempera-ture was decreased to 200°C, there was no loss, but the

quality of purification deteriorated significantly

Apparently, the loss of mono- and dibrominated

diphenyl ethers is associated with the problem of

desorption rather than with chemical transformations

This hypothesis was confirmed by elution with

meth-anol, when MoBDE and DiBDE were desorbed

quantitatively However, this elution method has no

practical significance, since methanol dissolves

alu-mina, which precipitates from solution upon

precon-centration Successive elution of the column with a

mixture of hexane–methanol (19 : 1, vol) and

mix-tures of hexane–DCM–methanol in volume ratios of

17 : 1 : 2, 8 : 1 : 1, and 10 : 9 : 1 (hereinafter, each

frac-tion of 20 mL) did not achieve quantitative elufrac-tion of

MoBDE When eluted with pure toluene, zero

recov-eries were obtained for MoBDE and DiBDE;

isopro-panol eluted less than 50% Quantitative desorption of

all PBDEs was achieved using a hexane–diethyl ether

mixture (4 : 1, vol) A mixture containing two times

less ether eluted at least 70% of low-brominated

PBDEs, and the rest analytes were eluted

quantita-tively

Two-stage purification Fractionation is an

essen-tial tool for fine purification, but nonselective

adsorp-tion barely enables the quantitative separaadsorp-tion of trace components from the main components of the matrix

It was noted earlier that a combination of fractionation and chemical purification is a more effective approach The use of silica impregnated with sulfuric acid is a conventional method of removing macro amounts of fat and many other extractable compo-nents of the matrix in the determination of PCBs and other substances that withstand such effects, but the tarring or saponification of organic substances under the effect of sulfuric acid leads to the “sticking” of the column, because of which the rate of passage of the solvent decreases In addition, the reaction products have uncontrollable adsorption properties, which complicates the work, leads to the loss of analyte sub-stances, and increased solvent consumption We used

a different approach, namely, the binding of the gross amount of fatty acids with potassium silicate and Flo-risil (magnesium silicate) Despite the similar nature

of these substances, they are likely to bind different components of the fat matrix in different ways The highest efficiency was shown by a column containing

a layer of Florisil between two layers of potassium sil-icate, separated by a layer of anhydrous sodium sul-fate, and a layer of silica impregnated with sulfuric acid in the lower part of the column to remove residual components of the matrix Samples in 5 mL of hexane were applied to a dry column; the substances to be determined were eluted with 50 mL of hexane The eluate was fractionated in a neutral alumina column The solution can be applied on a column with alumi-num oxide without preconcentration or by evaporat-ing it up to 2–3 mL in a rotary evaporator The purifi-cation procedure and the amount of adsorbents and solvents are shown in Fig 1 The results of the deter-mination of PBDEs in a sample of fish oil are given in Table 2 Each purified extract was analyzed twice using the long and short chromatographic columns The recovery rates of all PBDE congeners in fish oil samples were not less than 83%; only in the blank experiment, lower values were obtained for BDE-3 and BDE-197 The results are characterized by excel-lent reproducibility in determining both the concen-trations of native PBDEs and the recovery rates (Table 2), which demonstrates the reliability of the proposed method High recovery rates make it possi-ble to decrease the consumption of solvents, which is

110 mL; this is more than four times smaller than when using the FMS Total-Prep automatic sample preparation unit Contamination of the blank sample

is comparable to the values obtained with the use of the FMS unit, which is acceptable for moderately con-taminated samples but yields distorted results in the case of low concentrations of PBDEs

Problem of the blank experiment In the case of

PBDEs and PCBs, the problem of the blank experi-ment cannot be solved entirely, unlike PCDD/PCDF, when, at least for congeners that make a significant contribution to the total equivalent toxicity [26], a

Fig 1 Purification of fat-containing extracts in the determination of PBDEs with the degrees of bromination from 1 to 10

~0.5 g of fat in ~5 mL of hexane

Column to remove fat (internal diameter 14–15 mm)

Na2SO4 ~ 2 g

K2SiO3 ~ 4 g

Na2SO4 ~ 1.5 g Florisil ~ 4.5 g

K2SiO3 ~ 4 g

Na2SO4 ~ 1.5 g

H2SO4 /SiO2(30%) ~ 1 g

Fractionation on neutral alumina

(column with internal diameter 9 mm)

4 g, activated at 400°C for 16 h, conditioning with 15 mL of hexane

Washing with 15 mL of hexane

(waste)

Without preconcentration

or down to 1–2 mL

Elution with 50 mL

of hexane

Elution with 20 mL of a diethyl ether–

hexane mixture (1 : 4, vol)

Low- and medium-brominated congeners, SGE HT-8 or SGE-5 column 25–30 m (or equivalent)

Preconcentration to ~10 µL

or GLC−MS analysis

High- and medium-brominated congeners, J&W DB-5ht column 10–15 m

vanishingly small level of the blank experiment can be

achieved

Sources of PBDEs and PCBs in the blank sample

are of a universal nature; therefore, solutions to the

problem are also similar We should consider all

adsor-bents, solvents, glassware, synthetic polymer

materi-als, and even air in the laboratory potential sources of

contamination The contribution of each source var-ies, depending on the qualification of the adsorbent or solvent, and may vary from batch to batch The contri-bution to the contamination of solvents of “pesticide grade” qualification or intended for the determination

of PCDD/PCDF and PCBs is usually very low, but it

is reasonable to minimize their consumption, and not

only for economic reasons You should also pay

atten-tion to the selecatten-tion and preparaatten-tion of adsorbents,

abandon plastics in contact with solvents, and calcine

glassware It is also necessary to ensure the purity of

extractors [27] and reusable glassware, which is

desir-able to be silanized [28] All these measures, even

when transferring sample preparation to an isolated

room with a particular air purification system, do not

guarantee a complete solution to the problem of the

blank experiment [29]

We did not set the goal of achieving ultralow limits

of detection and limited ourselves to minimizing the

inf lux of contaminations from the two main sources,

which turned out to be silica and Florisil Because of

the specificity of the physicochemical properties of

sil-ica, it is almost impossible to remove impurities

with-out deteriorating its adsorption properties, so the

solu-tion was to replace convensolu-tional silica gel 60 with a

high-purity analogue For the purification of Florisil,

we performed ASE with isopropanol and hexane at

180°C In both cases, the level of contamination

decreased several times but remained higher than

required The best result is the calcination of Florisil in

a muff le furnace at 595°C for 12 h; however, this

increases its adsorption activity, which leads to a sharp

drop in the recoveries in the blank experiment Adding

5% of DCM to hexane during column elution solved this problem The results obtained when analyzing the three fat matrices and the blank sample (Table 3) show that we managed to decrease the level of contamina-tion of the blank sample, lowering the determinacontamina-tion limits, while the recovery rates remain consistently high

The developed method is intended for determining PBDEs in samples of feed and food containing approximately 0.5 g of fat; however, the techniques used in it enable the method to be used for analyzing larger weights or for analyzing other matrices with minor modifications and for determining other sub-stances, for example, PCBs, organochlorine pesti-cides, or polycyclic aromatic hydrocarbons (by excluding silica impregnated with sulfuric acid from the purification setup) In the proposed method, rela-tively small amounts of adsorbents and only 110 mL of solvents are spent for purifying one sample The pro-cedure can be carried out without the use of chlori-nated organic solvents, and consistently high recover-ies show the potential of optimizing the cost of analysis

in the future

Table 2 Concentrations and recoveries (Rex ) of polybrominated diphenyl ethers in the analysis of fish oil samples

* Recalculated to a weighed portion of 0.5 g.

** Reference compounds added after sample preparation to control recoveries.

Analyte

c, pg/g Rex, % c, pg/g Rex, % c, pg/g Rex, % c Rex c, pg/g* Rex, %

The authors are grateful to the Center for

Ecosys-tem Safety at the Department of Biology of the

Mos-cow State University for technical support

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Translated by O Zhukova