Environ. Sci. Techno/. 1995,29, 1686-1690
Det-on
of Stmls in
DANG-KHOA NGUYEN,’ A U G U S T E BRUCHET,’ A N D PATRICK ARPIN 0 * I *
Centre International de Recherche Sur 1’Eau et I’Environnement (CIRSEE), Socidtd Lyonnaise des Eaux, 38 rue du Prdsident Wilson, F-78230 Le Pecq, France, and Laboratoire de Chimie Analytique, Institut National Agronornique, 16 rue Claude Bernard, F- 75231 Paris, France
Sterols were quantitatively extracted from freezedried sewage sludges with high (60-80%) organic matter contents by simultaneous supercritical fluid extraction and BSTFA derivatization. The sterol trimethylsilyl derivatives were finally analyzed by capillary GC/MS. This protocol yielded similar results but was more rapid than the U S EPA method of ultrasonic extraction with a (1:l) methylene chloride/acetone mixture, followed by trimethylsilylation and GC/MS. Sample preparation by combined TMS/SFE and quantitative analysis by GC/MS were applied to the determination of several free sterols in sewage sludges from four different origins. Coprostanol, a fecal sterol, was present in all municipal wastewater digested sludges at the highest concentration, ranging from 2.28 to 4.05 mg/g, and accounted for 38-62% of the total detected sterols.
Introduction Significant amounts of sterols arising from household activities are regularly discharged into municipal wastewaters. Coprostanol and cholesterol, the major sterols found in raw wastewaters, have been detected at high concentration levels of 250 and 190 ,ug/L, respectively (1, 2). Sterols are poorly soluble in water (e.g., cholesterol water solubility is cu. 19 ppb at 30 “C (311, thus they tend to associate with particulate matter and to accumulate in sewage solids from primary wastewater treatment. These sterols, in particular coprostanol (a sterol formed by the bacterial reduction of cholesterol in the intestine of higher animals), are resistant to anaerobic biodegradation and will therefore be present in the digested sludge at the end of the treatment process. This sewage sludge is sometimes disposed to agricultural land resulting in a possible source of fecal pollution in the environment, which can be traced by the presence of coprostanol. Sterols are frequently extracted from sediments or sludges by liquid solvent extraction (4,geither in a Soxhlet apparatus (2, 6)or by sonication as recommended by the American Environmental Protection Agency (US.EPA) ( 7 ) . These extraction processes are generally time consuming and often require an extract cleanup step. Furthermore, small amounts of sterols can be lost during the cleanup and extraction process, especiallyby adsorption on Soxhlet filter cartridges (2). In general, final sterol quantitation is achieved either by GC or GUMS of their trimethylsilylether (TMS) derivatives, thus an additional time is also required for the derivatization step. This conventionalmultistep protocol for sterol extraction and derivatization can be more efficiently replaced by a new one-step method that combines in situ trimethylsilyl derivatization, using N,0-bis(trimethylsily1)trifluoroacetamide (BSTFA),and COz supercritical fluid extraction (SFE). The extracted sterol TMS derivatives are ready to be next quantified by GUMS. We have compared two extraction methods-the twostep ultrasonic extraction and TMS derivatization (US TMS), according to U.S. EPA method 3550A ( 7 ) , and the new one-step method of trimethylsilylation/supercritical fluid extraction (TMS/SFE)-for the determination of several free sterols in sewage sludges. We also report on the application of the TMS/SFE method to the investigation of digested sludgesfrom four municipal wastewater treatment plants that lead to the identification and determination of 14 sterols.
+
Experimental Section Sample Origin and Preparation. Digested sludges were collected from four French municipal wastewater treatment plants in the Lyon (SaintFons) and the Paris areas (AsniBres/ Oise,AchBres, and Evry). Sludge samples were centrifuged at 15000 rpm for 20 min, the supernatant was discarded, and the pellets were dried by lyophilization. Reagents. Sterol standards and coprostanol (analytical grade) were obtained from Sigma and Serva, respectively. * Corresponding author; Fax: ( f 3 3 ) 1-44-08-16-53. Centre International de Recherche Sur 1’Eau et I’Environnement (CIRSEE). + Laboratoire de Chimie Analytique. +
1688 ENVIRONMENTAL SCIENCE &TECHNOLOGY I VOL. 29, NO. 6,1995
0013-936W95/0929-1686$09.0010
@ 1995 American Chemical Society
NO-Bis(trimethylsily1)trifluoroacetamide with 1%of tri-
+
methylchlorosilane (BSTFA 1%TMCS) was purchased from Pierce. Twice distilled acetone and pestipure-grade methylene chloride were obtained from SDS, France. Benzo[b]fluoranthene (internal standard) was obtained from the Community Bureau of Reference (BCR, Brussels, Belgium). Ultrasonic Extraction. Sonication extraction was performed according to the EPA (Environmental Protection Agency) sonication extraction method (U.S. EPA Method 3550A) (7) using a Vibracell system (purchased from Bioblock Scientific, Illkirch, France) that includes a 375-W sonicator ultrasonic processor and a cell disruptor sonic probe. The ultrasonic disruptor was operated as recommended by the US.EPA, i.e.,with energy powered on during 50% of total time. A 20-mg dried sludge sample was ultrasonically extracted for 5 min with 5 cm3 of acetone/ methylene chloride (l:l, vv) solvent mixture. After extraction, the suspension was centrifuged at 15000 rpm for 15 min, and the solvent supernatant was decanted. The entire procedure was repeated three times with fresh solvent, and the three extracts were combined and concentrated to dryness under a flow of nitrogen. Sterols in the dried extract were derivatizedto their trimethylsilylethers at 70 "C during 20 min, using 1 cm3 of BSTFA in methylene chloride (1:2). The solution was spiked with 8 pg of the internal standard (benzo[b]fluoranthene),and 1pL was directly injected into the capillary GUMS. Combined in Situ Derivathtion-Supercritical Fluid Extraction. A 20-mg dried sludge was weighed on a Whatman GF/F filter cut to about 2.5 cm diameter. The filter was introduced in a 7 cm3 extraction cell, two glass wool plugs were placed on the top and the bottom of the extraction cell, and 1 cm3 of BSTFA in acetone (1:2) was added into the cell. Two additional layers of Whatman GF/F filter, cut to the extraction thimble diameter, were placed on top and bottom of the thimble cap to avoid frit plugging. SFE was performed using a Hewlett-Packard Model 7680T supercriticalfluid extractor and carbon dioxide (SFC grade CO2 from Alphagaz, France) at a density of 0.8 g/cm3 (corresponding to a pressure of 36.5 MPa) and a flow rate of 3.0 cm3/min. One step of static extraction during 15 min followed by a second 15-min step of dynamic extraction were carried out with the extraction chamber temperature kept at 80 "C. Sample extracts were collected on an octadecyl-bonded (ODS) silica cartridge, kept at 30 "C during the extraction and rinsing stages. The ODS cartridge was successively eluted by two 1.0 cm3 volumes of methylene chloride. The second fraction was to control that sterols were completely recovered by the first extraction. The first fraction containing all trimethylsilylsterol derivatives was spiked with 8 pg of the internal standard (benzo[blfluoranthene) and analyzed by capillary GUMS. Combined Gas ChromatographylMass Spectrometry. GClMS was accomplished with a Delsi, Model DN200 capillary gas chromatograph, coupled to a Unicam quadrupole mass spectrometer, Model Automass 50. A J&W DB-17narrow bore capillary column (30 m x 0.25 mm i.d. with 0.25pm film thickness) was eluted with cu. 1 cm3/min of helium. The column was connected at one end to the GC on-column injector via a 2 m x 0.32 mm i.d. retention gap made of deactivated fused-silica tubing; the other end was directly introduced into the ion source of the mass spectrometer. The initial oven temperature of 50 "C was held for 1 min. increased to 230 "C at 10 "Clmin and then
absolute response facton
(arbitrary units)
I500
I300
lZA
mlz 484
2400 d z 386
'A
1500
mlz 411
~
3500 r d z 458
1300
I
mlz 460
8W0
32
'A 'A
mlz 370
38
35
41
T h e (min)
FIGURE 1. GWS analysis of a calibration solution containing equal amounts of authentic sterols. Computer-generated selected ion current profiles for the [MI'+ or [M - MI'+ ions of the sterol TMS derivatives: (1) coprostanol, (2) epicoprostanol, (5) cholestanol, (6) cholesterol, (9) campesterol, (10) ergosterol, (11) coprostanone, (12) stigmasterol, (13) stigmastanol, and (14) sitosterol.
to 290 "C at 3 "C/min, where it was held for 5 min. Acquisition and processing were accomplished with the Lucy Data System, Version 2.10. The MS source temperature and pressure were 120 "C and Pa, respectively. Filament emission current was 305 mA, and 70 eV electron impact mass spectra were acquired by repetitive scanning between mlz 50 and 550 in 1 s. QuantitativeDeterminations. Quantitative measurements of the extracted sterol TMS ethers were achieved by GUMS. Calibration solutions of the authentic sterols were prepared at concentration of 10 mg/L per pure steroidal mono-alcohol. The sterols were derivatized to their TMS ethers, and the final solution was spiked with the internal standard. An example is presented (Figure 1) showing the computer-generated ion current profiles, from full-scan data acquisition, that are selective for the investigated sterol TMS ethers. Four sterols (24-methylcoprostanol,24-ethylcoprostanol, 24-methylepicoprostanol,24-ethylepicoprostanol) were quantified by using the response factors of coprostanol and epicoprostanol, respectively, because authentic standards could not be obtained. Selected ion current profiles were used to limit interferences by possible coeluting compounds. The [MI'+ molecular ion was sufficiently abundant to be used for cholestanol (mlz = 460), cholesterol (mlz = 458), campesterol (mlz = 472), ergosterol (mlz = 468), coprostanone (mlz = 386), stigmasterol (mlz= 484), stigmastanol (mlz = 4881, and sitosterol (mlz = 486). For the other sterol TMS derivatives, the [M - 90]*+fragment ion was selected, corresponding to the neutral loss of a TMS(0H)group from the molecular ion. This was the case for coprostanol and VOL. 29, NO. 6,1995 / ENVIRONMENTAL SCIENCE &TECHNOLOGY
1687
absolute response factors (arbitrary units) I8000
TABLE 1
1
nc
6C/MS Detennisation of Sterols from Achbres Digested SludBe by Consecutive Ultrasonic hactioa/TMS Derivatizatimi (US TMS) and by in Situ Supercritical Fluid Extraction/TMS Derivatization (SFUMS)
A
A
1400
+
Is
1
I
B sterol coprostanol epicoprostanol 24-methylcoprostanol 24-methylepicoprostanol cholestanol cholesterol 24-ethylcoprostanol 24-ethylepicoprostanol campesterol ergosterol coprostanone stigmasterol stigmastanol sitosterol total
I8000 I
C
nc~
1400
IS
1
I
1 32
5+6
' 2 -
D
I
I\
~~
34
_______ 36 Time (min)
38
40
FIGURE 2. Total ion current traces from the GCMS analysis of the sterol TMS ethers extracted from Achbras sewege sludge samples, usingtwo ditferentextreetionprocedures.PanelsAasd B: Combined in siru supercritical fluid eKtnction/trinrethyllyl~ion.Panels C and D Ultrasonic extractionfoilowed by trinwthylrilylation. (Panel A) Sterols in the first SFE extract (panel B) sterols in the second SFE extract; (panel C) sterols in the combined fraction from three successive ultrasonic extractions; (panel D) sterols in the fourth ultrasonic extract (1) coprostanol, (2) epicoprostanol, (3) 24methylcoprostanol,(4) 24-methylepicoprostanol,(5) cholestanol, (6) cholesterol, (7) 2 4 - e t h y l c o p r ~ o l (8) , 24-ethylepicoprostsnoI, (9) campesterol, (10) ergosterol. (11) coprostnnone, (12) stigmasterol, (13) stigmastanol, (14) sitosterol, and (IS) benzdblfluoranthene (internal standard). epicoprostanol (both at m/z = 370),24-methylcoprostanol
and 24-methylepicoprostanol(both at mlz = 3841, and 24ethylcoprostanol and 24-ethylepicoprostanol(both at mlz = 398).
Results and Discussion Sterol Recovery by the Two Methods. Since the true concentration of native sterols in sludge samples was a priori not known, one sample (Achkres)was exhaustively extracted using several consecutive extractions. After each step, the amount of recovered sterols was determined, and the extraction was repeated until the detected sterols were at the lower GClMS detection limit (0.5 pg/g). One sludge sample was successivelyextracted four times by combined SFE/TMS; however, traces of sterols (c5 pg/ g) were found in the second extract (Figure 2), and none in the following extracts, suggesting that extraction of the sterol TMS ethers was nearly complete after the first run. The same sample was also successively extracted four times by ultrasonic extractionwith aliquid solvent mixture, followed by trimethylsilylation. Sterol abundances decreased in the recovered fractions until the third ultrasonic extract. Negligible sterol abundances were found in the f o u a extract and were in comparable amounts with those found in the second SFE extract (Figure 2). Thus one-step of SFE is as efficient as three consecutive ultrasonic extractions. Total sterol recovery by both 1688 ENVIRONMENTAL SCIENCE & TECHNOLOGY I VOL. 29. NO. 6, 1995
concentration mg/Q of dried matter US TMS' SFVTMSb ( n = 3) ( n = 3)
+
4.10 f 0.35 0.96 f 0.15 0.20 f 0.05 0.08 f 0.02 0.41 f 0.01 0.45 f 0.05 0.88 f 0.09 0.19 f 0.04 0.05 f 0.01
