# Estrogenic Compounds (5044)

​ESTROGENIC COMPOUNDS
FORMULAE: Table 1
MW: Table 1
METHOD: 5044, Issue 1
CAS: Table 2
RTECS: Table 2
EVALUATION: PARTIAL
PROPERTIES:
OSHA: Table 2 NIOSH: Table 2 ACGIH: Table 2 Synonyms:
5044
Issue 1: 15 March 2003
Table 1
Table 2
SAMPLING
MEASUREMENT
SAMPLER:
FILTER, PTFE (37-mm, 2-:m)
TECHNIQUE:
HPLC, UV detection
FLOW RATE:
1 L/min
ANALYTE:
See Table 1
VOL.-MIN: -MAX:
150 L 1000L
EXTRACTION:
Methanol, extraction overnight at room temperature
SHIPMENT:
Ship at ambient temperature.
INJECTION VOLUME:
SAMPLE STABILITY:
30 days at 25 °C [1]
BLANKS:
2 to 10 field blanks per set
ACCURACY RANGE STUDIED:
Not determined
BIAS:
Not determined
OVERALL PRECISION(S r T ):
Not determined
ACCURACY:
Not determined
25 :L
MOBILE PHASE:
60% acetonitrile / 40% water @ 26 °C, 1.75 mL/min
COLUMN:
C18 reversed phase, 150 x 4.6-mm, 5-:m; in-line pre-filter, 2.0- :m
DETECTOR:
UV/Vis at 200 nm and 237 m:
CALIBRATION:
Standards in methanol
RANGE:
$-Estradiol 0.3 to 44 :g/sample Estrone 0.2 to 64 :g/sample Progesterone 0.5 to 64 :g/sample $-Estradiol 3-Benzoate 0.5 to 64 :g/sample [1]
ESTIMATED LOD: Table 3 PRECISION (S r ):
$-Estradiol 0.040 Estrone 0.039 Progesterone 0.030 $-Estradiol 3-Benzoate 0.032 @ 0.5 - 64 :g/sample [1]
APPLICABILITY: This method has not been used to evaluate field air samples. The method is based on a preliminary investigation analyzing field wipe samples collected during a health hazard evaluation at a facility producing birth control pills [1]. Appendix 1 contains information on recovery of analytes from wipe samples. INTERFERENCES: Any compound that elutes at the same HPLC retention times may interfere. OTHER METHODS: No validated methods were found.
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ​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.
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 ​EST RO GE NIC C OM PO UN DS: M ETH OD 5044, Issue 1, dated 1 5 Ma rch 2003 - Page 3 of 6 12. Determine the desorption efficiency and recovery in the range of interest for each lot of filters used for sampling. a. Prepare four tubes at each of three levels plus three media blanks. b. Add known amounts of the analyte or analyte mixture in methanol to the filters. c. Allow to stand $2 hr for solvent evaporation. d. Prepare sam ples (steps 6 through 1 0) and ana lyze together with standards (steps 15 and 16 ). e. D ete rm ine rec overy. 13. Analyze three quality control blind spikes and three analyst spikes to ensure that the calibration graph and re covery graph are in control.
MEASUREMENT: 14. Set HPLC according to manufacturer's instructions and conditions on page 5044-1. 15. Inject sam ple aliquot m anually or with autosam pler. 16. Measure peak areas.
CALCULATIONS: 17. Determine the mass in :g of analytes found on the sample filter, W , and on the average blank filter, B. 18. Calculate conce ntration, C, of analytes in the air volum e sam pled, V (L):
NO TE : :g/L / m g/m 3
EVALUATION OF METHOD: This method has been used to analyze field-generated wipe samples [2]. It wa s not evaluated with laborato rygenerated air samples. LOD/LOQ, extraction efficiency, sam pling stability and storage s tability studies were performed with laboratory-spiked filters. LOD/LOQ values (in :g/filter) for $-Estradiol were 0.8/2.5; for Estrone, 0.2/0.5; for Progesterone, 0.5/1.7; and for $-Estradiol 3-Benzoate, 0.5/1.7. Extraction efficiency for $-Estra dio l ra nged from 94 to 103% ; for E strone, 95 to 104% ; for Progesterone; 97 to 102% ; for $-Estradiol 3-Benzoate, 95 to 101%. An additional recovery study using W ash 'n Dri® wipes gave extraction efficiencies of 97 to 100%. The sta bility of the analytes on the filter m edia (s tatic efficiency) was evaluated by drawing air through spiked filters. Reco veries ranged from 94 to 102% for $-Estradiol; for Estrone, from 96 to 101%; for Progesterone, from 99 to 102% ; and for $-Estra dio l 3-B enzoate, from 92 to 101% . Reco very after 30-da y storage a t 25 °C was 93% for $-Estradiol, 94% for Estrone, 98% for Progesterone, and 111% for $-Estradiol 3-Benzoate.
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ​EST RO GE NIC C OM PO UN DS: M ETH OD 5044, Issue 1, dated 1 5 Ma rch 2003 - Page 4 of 6 REFERENCES: [1] [2] [3]
Mathews ES and Neum eister CE [2000]. Backup Data Report for Method 5044, Estrogenic Compounds by HPLC (unpublished), NIOSH, DART. Neum eister CE [1983]. Analytical Report for Sequence 1400. Cincinnati, OH: National Institute of Oc cup ationa l Safe ty and H ealth (u npu blishe d). Sixth Annual Report on Carcinogens [1991]. USDHHS/PHS, National Toxicology Program
METHOD WRITTEN BY:
Elaine S. Mathews and Charles E. Neumeister NIOSH, DART
TABLE 1. PROPERTIES Compound
Formula
M.W.
M.P. (O C)
UV Max (nm)
$-Estradiol
C18 H24 O2
272
173-179
206
Estrone
C18 H22 O2
270
254-256
198
Progesterone
C21 H30 O2
314
127-131
237
$-Estradiol-3-Benzoate
C25 H28 O3
376
191-196
201
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ​EST RO GE NIC C OM PO UN DS: M ETH OD 5044, Issue 1, dated 1 5 Ma rch 2003 - Page 5 of 6
TABLE 2. GENERAL INFORMATION
Compound
$-Estradiol
Synonyms
dihydroxyfollicular hormone
CAS
RTECS
Exposure Limit
50-28-2
KG2975000
None Specified
53-16-7
1009137ES
None Specified
57-83-0
None TW 0175000 Specified
50-50-0
KG4050000
dihydroxyestrin oestra-1,3,5(10) triene-3,17-betadiol
Estrone
3-hydroxyestra-1,3,5(10)-trien-17-one 1,3,5-estratrien-3-ol-17-one oestrone folliculin
Progesterone
pregn-4-ene-3,20-dione luteohormone Corlutin
$-Es tradiol-3-Benzo ate estra diol benzoa te oes tradiol mono ben zoate (17$)-estra-1,3 ,5(10)-triene-3,17-diol 3-benzoa te Be novocylin
TABLE 3. LOD/LOQ DETERMINATION
Compound
LOD
LOQ
    g/filter
    g/filter
$-Estradiol
0.8
2.5
Estrone
0.2
0.5
Progesterone
0.5
1.7
$-Es tradiol 3-Be nzoa te
0.5
1.7
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition
None Specified ​EST RO GE NIC C OM PO UN DS: M ETH OD 5044, Issue 1, dated 1 5 Ma rch 2003 - Page 6 of 6 APPENDIX 1: PREPARATION OF SPIKES ON W IPES: W ipe m edia (W ash 'n Dri® Moist Dispos able Tow elettes) were opened and spread to evaporate to dryness. Dried towe lettes w ere rolled and ins erted into 16 x10 0-m m PT FE -lined s crew ca p tubes. A Ham ilton syringe was used to place measured am ounts of stock solution in methanol (50 :L containing 40 :g of each analyte) on each wipe. After allowing the spikes to evaporate, 8 mL of m ethanol was added to each tube, and the tubes were capped and rotated overnight at room temperature to extract the spiked analyte from the wipes.
EFFICIENCY OF EXTRACTION: Com pound
Efficiency of Extraction (%) (n=6)
Sr
$-Estradiol
100
1.4
Estrone
97
3.1
Progesterone
99
0.82
$-Es tradiol 3-Be nzoa te
99
0.76
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition