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EST RO GE NIC C OM PO UN DS: M ETH OD 5044, Issue 1, dated 1 5 Ma rch 2003 - Page 2 of 6 EQUIPMENT:

REAGENTS: 1. 2. 3. 4. 5. 6. 7. 8.

9.

W ater, distilled, deionized, degassed Aceton itrile, HPL C grade , degass ed.* Me than ol, HP LC grad e.* $-Estrad iol (Sigm a E8875 o r equivalen t)* Estrone (Sigm a E975 0 or equivalent)* Prog estero ne (Sigm a P0130 o r equivalen t)* $-Estradiol 3-Benzoate (Sigma E8515 or equivalent)* Sto ck sta ndard solution s: p lace approxim ate ly 40 mg of each analyte, weighed to 0.001 mg, in separate 20-m L vials and dissolve in 20-mL m eth anol. Estrogen mixed standards: combine 4 mL of eac h stock standard solution in a 20-m L vial. Prepare serial dilutions of this m ixtu re in methanol to 0.05 µg/mL.

1.

2 3. 4. 5. 6. 7. 8. 9. 10.

See SPECIAL PRECAUTIONS

11.

Filters: PTFE-laminate, 37-mm , 2-:m pore size (SKC Inc., Cat No. 225-17-07 or equivalent), cellulose spac er ring, 37-mm OD, 32-mm ID (SKC Inc., Cat. No. 225-23 or equivalent) in a 37-mm cassette filter holder. Culture tubes, PTFE-lined screw cap, 16-m m x 100-m m . Vials, auto-sam pler, 4-mL, with PT FElined septa. Pipets, volumetric (0.5- to 20-mL) Vials, PTFE-lined screw cap, 20-mL. Forceps. Ham ilton syringes, 50-:L and 100-:L. Syringe, disposable, 10-mL. Syringe filter, PTFE, 0.45-:m HP LC with integ rator and autosam pler; UV/Vis detectors in series (200-nm and 237-nm ); C18 colum n, 150x4.6-m m (Alltim a; Alltech As soc iates In c., State Co llege P A, C at. No . 88053, or equ ivalent); in-line pre-filter, 2.0 :m (Opti-So lv; Op timize Tec hnologies, or equivalent). Rotator, rotating tube shaker (LabquakeThe rm olyne or equivalent).

SPE CIAL PR ECAU TIO NS: Ac eto nitrile and m eth anol are flam m able and health hazards. Meth anol is a cumulative poison. Estrogenic compounds m ay be carcinogens [3]. Handle in a well ventilated hood and wear protective clothing. SAM PLING : 1. 2. 3. 4.

Calibrate each personal sampling pump with a representative sampler in line. Take personal samples at 1 L/min for a total sample size of 150 to 1000 L. Pack filter cassettes sec urely for shipment (unrefrigerated). Sto re sam ples at am bient te m perature upon re ceipt at the laborato ry.

SAMPLE PREPARATION: 5. 6. 7. 8. 9. 10.

Rem ove each filter from the cassette holder, roll with forceps and place in a 16 x 100 mm culture tube. Add 4.0 mL methanol to each tube. Prepare m edia and reage nt blanks in the sam e m anner. Cap each tube tightly and place on rotator. Rotate $8 hr at room temperature. Filter extracts, if necessary, using a disposable syringe fitted with a 0.45-:m PT FE filter into a clean vial. Transfer all extracts to labeled autosampler vials.

CALIBRATION AND QUALITY CONTRO L: 11. Ca librate daily with at leas t six wo rking standa rds over the ra nge of interest. a. Prepare serial dilutions of the stock mixture in the range of 0.08 to 80 :g/m L b. Analyze with sam ples and blank s (steps 15 an d 16). c. Pre pare a calibration g raph (pea k area vs m ass of an alyte, :g per sam ple).

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition