FINAL REPORT The Use of Coprostanol as an Indicator of the Sanitary Quality of New Jersey Waters Year 3 Contract Number: SR 00-049 Jae Kwak and Joseph D. Rosen Department of Food Science Rutgers University May 28, 2002 Submitted to Tom Atherholt Division of Science, Research and Technology New Jersey Department of Environmental Protection 1 TABLE OF CONTENTS Executive Summary 3 Introduction/Problem Statement 3 Project Design and Methods 4 Overall Plan 4 Separation of Analyte(s) from Water 5 Derivatization 5 Mass Spectra of TMS-coprostanol and TMS-cholesterol 5 Mass Chromatography 5 Electron Ionization or Chemical Ionization 6 Linearity, Surrogate 6 Internal Standard 7 Analytical Procedures (Extraction; GC/MS) 7-8 Microbial Analysis in Water Samples 8 Coprostanol Analysis in Food 9 Microbial Analysis in Food 10 Estrogenic activity of sterols and water sample 11 Quality Assurance 11 GC/MS 11 Data Quality Requirements: 13 Standard curves for coprostanol and cholesterol-2,2,4,4,6-d5 13 Quality Control Checks, Documentation 14 Data Validation, Performance and System Audits 14 Results and Discussion 15 Recovery Results Using a DB-17 Chromatography Column 15 Sensitivity Determination Using a DB-17 Chromatography Column 15 Analytical Results for Well Water Samples 15 Analysis Results for Sewage Samples 16 Sensitivity of Chemical vs. Microbial Method 16 Coprostanol in Food 16 Estrogenic activity of sterols and water sample 17 Effect of water treatment on concentration of steroids 17 Conclusions 17 References 17 Presentations and Publications 18 Appendix 18 Figure Legends 19 Table 1 20-28 Table 2 29-30 Tables 3 &4 31 Figures 32-41 2 EXECUTIVE SUMMARY This report covers the period between July 1, 1998 and May 28, 2002. An analytical method for the determination of coprostanol in water has been developed. In brief, the method consists of passing four liters of water (spiked with internal standard cholesterol-d5) through a 50 mm diameter C-18 disk, eluting the disk with solvents, evaporating, derivatizing with bis-trimethylsilyltrifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane, evaporating to 250 mL and then injecting 2 mL onto a DB17 column connected to a Finigan Ion Trap Mass Spectrometer operated in the EI mode with quantitative analysis made by mass chromatography. Sensitivity of the method was between 2 and 10 parts per trillion for 4L and 1L samples, respectively. Using this method, a total of 141 samples (126 well water samples, 8 sewage water samples and 7 field blanks) were analyzed. Only two of well water samples contained coprostanol (0.013 mg/L and 0.0035 mg/L). No coprostanol was found in the other well water samples. Forty-one of the well water samples contained other sterols (4 definitively- identified sterols: cholesterol, campesterol, stigmasterol, sitosterol, 5 tentatively identified sterols: 24-ethylcoprostanol, brassicasterol, 22-dehydrocholesterol, fucosterol, isofucosterol, and two unidentified sterols. These sterols, as well as coprostanol, epicoprostanol, and campestanol were found in sewage samples at much higher levels. Coprostanol concentrations in sewage water ranged between 4 mg/L and 2,136 mg/L. Cholesterol, campesterol, stigmasterol, and sitosterol were positive for beta receptor activation, an indicator of endocrine disruptor activity. Sensitivity of the chemical method was compared to the standard coliform assay by analyzing a water sample collected from the Raritan River. Total coliforms and fecal coliforms in the river water were determined to be 15 CFU/mL and 9.3 CFU/mL, respectively. The microbial method was much more sensitive than the chemical method when the same volume was analyzed. Coliforms were detected from only 0.1 mL of the river water sample, while at least 50 mL was required to definitely detect coprostanol by the chemical method. An attempt was also made to use the analytical method for the determination of coprostanol in food as an indicator of fecal contamination before ionizing radiation was applied. We were able to detect coprostanol in both chicken and in ground beef. However, we were also able to detect coprostanol in beefsteak, suggesting that the cow is capable of metabolizing cholesterol to coprostanol, thus making the assay unusable for our desired purpose. INTRODUCTION/PROBLEM STATEMENT The sanitary quality of drinking water and waters used for bathing and water sport activities is monitored using bacterial indicator organism tests such as those for total coliforms, fecal coliforms or enterococcus bacteria. These tests may not always be valid indicators of the possible presence of fecal pathogens since viruses, Giardia and 3 Cryptosporidium are more resistant to chemical disinfection and a variety of environmental stresses than the stated indicator bacteria. Therefore, in chlorinated waters, as well as other situations, pathogenic viruses and protozoa may be present in the absence of indicator bacteria. Also, the tests for indicator bacteria take 24 hours for initial results and 48 to 72 hours for confirmatory results. To better protect the public from unsafe waters, faster, more reliable test methods are needed. The purpose of this project was to examine the usefulness of coprostanol as a reliable, chemical indicator of fecal pollution. Coprostanol is a product of the microbial transformation of cholesterol in the intestines of higher animals. Current analytical methods for coprostanol are complex, but the potential exists for the development of a simpler, quicker assay. This project had three objectives. One, to streamline current analytical procedure used to detect and quantify levels of coprostanol. Two, to measure levels of coprostanol and bacterial indicator organisms in a variety of fecal pollution sources and three, to measure levels of coprostanol and indicator bacteria in several environmental waters such as ground waters used for drinking and fresh and marine waters used for bathing, shellfish harvest, and water sport activities. PROJECT DESIGN AND METHODS Overall Plan The analysis consists of two major operations: 1. Separate the analyte(s), present in nanogram amounts from the water, which is present in kilogram amounts. 2. Use gas chromatograpy-mass spectrometry for separation from other components of the matrix as well as sensitive and selective detection. Separation of Analyte(s) from Water Until recently, solvent-solvent extraction was used to extract remove organic materials from water. The organic materials, being less polar than water, would preferentially associate with the solvent. The solvent and the analyte(s) would then be separated from the water because they are immiscible. While effective, this method requires large amounts of organic solvents (that have to be disposed of), the attention of the analyst and is time-consuming. Solid Phase Extraction (SPE) is a technique in which water is passed through material which will bind the analytes of interest while allowing the water to pass through. Of the several SPE adsorbents we evaluated, we found that octadecylsilane was best. In order to meet the sensitivity requirements, large volumes of water would have to be analyzed. Thus, instead of the usual SPE minicolumn, we decided to use the J.T. Baker SpeediskTM, which has the capacity to absorb analytes from large quantities of water using a flow rate of 200 mL per minute at 25” Hg. 4 Derivatization Sterols such as coprostanol or cholesterol do not elute from a gas chromatography column very well because of their relative polarity (Figure 1A), whereas the chromatography is much improved if they are first converted to materials of lesser polarity (Figure 1B). Such a conversion is shown in Figure 2: the polar hydroxyl group of coprostanol is reacted with a derivatizing agent such as trimethyl chlorosilane or bis- trimethylsilyltrifluoroacetamide (BSTFA) to form trimethylsilyl coprostanol (TMS- coprostanol). The major advantage of derivatization is a more symmetrical peak shape, which translates into better separation from other sterols and matrix impurities as well as improved quantification. The major disadvantage of derivatization is the introduction of an additional step which leads to a slower overall analysis time and an additional source of lower precision. In order to decide between the two possibilities, sensitivity comparisons were made between derivatized and underivatized sterol standards. The sensitivity for underivatized coprostanol and underivatized cholesterol were much less than TMS-coprostanol TMS- cholesterol in both the EI and CI modes. Mass Spectra of TMS-coprostanol and TMS-cholesterol The EI mass spectrum of TMS-coprostanol is shown in Figure 3. The molecular weight of this material is 460 amu but is too unstable to be recorded. The peak at 370 m/z is due to loss of HOSi(CH3)3; the peak at 355 m/z is due to loss of an additional methyl group; the peak at 257 m/z is due to loss of the C8H17 side chain; the peak at 215 m/z is due to cleavage of the five membered ring (loss of propylene). The EI mass spectrum of TMS- cholesterol is shown in Figure 4. In contrast to the coprostanol derivative, TMS- cholesterol does exhibit a molecular ion at m/z 458. The peak at 443 m/z is from loss of a methyl group; m/z 368 is due to loss of HOSi(CH3)3 from 458 and 353 is loss of another methyl group from m/z 368. The ion at m/z 329 is presumed to arise from loss of octane side chain from the 443 ion. The peaks at m/z 255 and 213 are analogous to the TMS- coprostanol peaks at m/z 257 and 215. The CI mass spectrum of TMS-coprostanol is shown in Figure 5. Chemical ionization is much gentler than electron ionization and this results in less fragmentation. In addition, protonated molecular ions are more commonly seen in CI than in EI. In the case of TMS-coprostanol, however, the expected protonated molecular ion at m/z 461 is not seen; instead there is a small ion at m/z 459.