Use of a New HPLC Stationary Phase for One-Step Clean-Up of Human Milk for PCDD, PCDF, and PCB Analysis Maria Jose Gonzalez: BegoAa Jimenez, and Luis Manuel Hernandez Department of Environmental Contamination, Institute of Organic Chemistry (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain

Claire Vidal-Madjar and Helen Place Laboratoire de Physico-Chimie des Biopolymere, CNRS, Universite Paris-Val de Marne, UM 27, 2 rue Henry Dunant, 94320 Thiais, France

Key Words: HPLC Stationary phase Internal surface reversed phase (ISRP) One-step clean-up Human milk PCDDs PCDFs PCBs

1 Introduction Identification and quantification of polychlorodibenzo-p-dioxins (PCDDs), polychlorodibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs) in complex biological matrices are very difficult because low levels of these compounds are present with a number of other organic components at much higher concentrations. As the complexity of the matrices increases, more sophisticated and tedious multistage sample clean-up procedures are required.

A one-step extraction and pre-cleaning procedure has been achieved for the separation of high molecular weight compounds (proteins, lipids, ...) from PCDDs, PCDFS, and PCBs in human milk samples. The HPLC Ultrabiosep column used as stationary phase makes it possible to reduce the analysis time, the number of steps, and losses of the most liposoluble isomers.

This new phase can be classified in the category internal surface reversed phase (ISRP) [ I ] : the external surface is modified with a biological polymer and the internal side is chemically bonded with a selective hydrophobic phase. Molecules too big to reach the inner surfaces, such as proteins and high molecular weight lipids, pass right through the ISRP without being retained.

2 Experimental HPLC was performed with a Perkin Elmer Series 10 pump, a 50 ~1 sample loop, and an LC-15 UV detector operating at 254 nm The mobile phase in isocratic mode was a 7 3 mixture of 0 067 M tampon phosphate (pH 7 4)-acetonitrile at a flow of 1 ml/min The HPLC Ultrabiosep column (15 cm x 4 6 mm i d ) and precolumn were obtained from SFCCiShandon Eragny, France

High resolution GC was performed with a Perkin Elmer Model 8310B chromatograph equipped with a 63N1 electron capture detector and fitted with a 50 m x 0 22 mm i d fused silica WCOT capillary column coated with BP-5 (SGE)

3 Results and Discussion Figure l a shows the separation of OCDD from the other high molecular weight components of a sample of human milk (50 p1) spiked with octachlorod!benzo-p-dioxin (500 ng), Figure lb shows the separation of a similar quantity of Aroclor 1260 from the

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Figure 1

Separation of a) OCDD and b) Aroclor 1260 from other components of human milk.

0 1993 Dr. Alfred Huethig Publishers - Journal of Hiah Resolution Chromatography 129

Short Communications

other milk components. Proteins, triglycerides, and other high molecular weight compounds were not retained. We have deter- mined the retention time of the 2,3,7,8-tetrachloro isomer, and the hexa- and heptachloro isomers of PCDD and PCDF, and the components of other commercial PCBs (e.9. Aroclor 1254); all have the same retention time as the OCDD.

We have to report that these compounds penetrate through to the inner surfaces of the stationary phase and are therefore chroma- tographically retained. The fraction corresponding to the organo- chlorine compounds was collected and, after purification with alumina a s reported previously [2], analyzed by HRGC with ECD to confirm the nature of the compounds and the 100 % recovery of the added standard.

The possibility of reducing the number of steps in the analysis of PCDD, PCDF, and PCB in human milk is very important. The first step, the isolation of the fat which contains the liposoluble compounds, must be exhaustive if the analysis is to be quantita- tive. It is usually performed by liquid - liquid extraction with methanol, sodium oxalate, diethyl ether, and petroleum ether [3-5]. The second step, elimination of the fat, is normally performed either by digestion with sulfuric acid [6-8] or by gel permeation chromatography on Biobeds SX 3 [4, 51. As the use of the new Ultrabiosep stationary phase replaces these two steps (extraction and pre-clean-up) with only one, use of the ISRP reduces not only the analysis time but also the possibility of losses in the pre-clean-up steps. The final clean-up on alumina (usually the third step in this analysis) will usually be sufficient for HRGC-HRMS analysis.

No changes in the performance and the pressure of the column were noticed during the experiments.

The results obtained here represent the first attempts to evaluate the ISRP column for the determination of PCDDs, PCDFs, and

PCBs human milk The method could be used without any modification for the levels of PCB isomers found in human milk (the ppb (ng/g) range) Levels of PCDD/F in human milk range from 3 to 300 ppt (pg/g) (from tetra to octa PCDDIF) relative to the amount of fat [3-81 As HRGC HRMS detection levels for tetra to octa PCDD/F isomers are in the range 0 1-1 pg per inlection into the system, it would be necessary to adapt the methodology for higher inlection volumes A 5 ml sample would contain enough PCDD/Fs to be detected bv HRGC-HRMS

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A Comparison of Formic Acid and Formamide as Modifiers of Super- critical Carbon Dioxide Compatible with Flame Ionization Detection John W. Oudsema and Colin F. Poole* Department of Chemistry, Wayne State University, Detroit, MI 48202, USA

Key Words: Supercritical fluid chromatography Packed columns Modifiers Formic acid Formamide

1 Introduction Favorable critical constants, safety, purity, cost, and compatibil- ity with flame-based detectors have made carbon dioxide the most widely used mobile phase in supercritical fluid chromato- graphy (SFC) [l-31. The limited solubility of polar compounds in supercritical carbon dioxide and its limited capacity to mask undesirable solute interactions with inorganic oxide-based sta- tionary phases are its principal disadvantages. Sometimes these

problems can be circumvented by converting the sample to a less polar derivative, thus rendering the analytes more soluble in the mobile phase and simultaneously modifying the structure of the analyte so that it is less likely to interact strongly with active sites in the stationary phase. An alternative means of overcoming these problems is the addition of modifiers to the mobile phase with the intention of

130 VOL. 16, FEBRUARY 1993 Journal of High Resolution Chromatography