Girls with PSD (n = 56), aged between 2 and 9 years, referred by their primary care pediatricians from all over the Republic of Macedonia, including the capital city of Skopje, were recruited following clinical evaluation at the Department for Endocrinology and Genetics and the Pediatric Clinic, Medical Faculty in Skopje. Blood samples were obtained at the time of diagnosis. The control group consisted of 24 normal healthy girls from different regions in the Republic of Macedonia, age-matched with the PSD group, who were evaluated at the Pediatric Clinic, for clinical conditions such as upper respiratory infections or pulmonary infections. Analysis for p,p’DDE was performed at the Toxicology Department of C.H.U. Sart Tilman in Liege, Belgium.

Concentration of p,p’-DDE in serum were measured using a gas chromatographic analyzer coupled to a tandem mass spectrometer detector. The following pesticides were also measured: DDT, lindane, arachlor, heptachlor, aldrin, endrin and hexachlorbenzene (HCB). Sample preparation included a liquid-liquid extraction (petroleum ether: diethyl ether; 98:2) followed by a solid phase extraction (Bond Elut Certify; Varian, Walnut Creek, CA, USA). The eluate was evaporated to dryness and reconstituted with the derivatization mixture [N, O-bis (trimethylsilyl)trifluoroacetamide (BSTFA)/trimethylchlorosilane (TMCS) 10% in n-hexane] before injection into the gas chromatograph (Saturn 2000; Varian). The column was an HP-5 Trace (30 m x 0.25 mm internal diameter) from Hewlett Packard (Wilmington, DE, USA). Ionization by electronic impact occurred at 70 eV. All solvents were pesticide-grade quality. Reference standards were obtained from Cambridge Isotope Laboratories (Andover, MA, USA). A calibration curve was constructed over the range from 0 to 40 ppb, and showed linearity. Endosulphan-d4 (0.5 ppb) was used as internal standard. The recovery of p,p’-DDE was 96%. The limit of detection (0.1 ppb) was defined as three times the standard deviation (SD) of the results from the lowest quality-control serum pool over the course of the analyses (n = 15). For p,p’-DDE, this was consistent with a detection limit of 0.1 ng/mL serum. The coefficients of variation were between 4.6 and 7.8%.