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Determination of human pharmaceuticals in surface water using solid-phase extraction and high-performance liquid chromatography-mass spectrometry

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Determination of human pharmaceuticals in surface water using solid-phase extraction and high-performance liquid chromatography-mass spectrometry
Creator:
Cahill, Jeffery Dene
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English
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112 leaves : illustrations ; 28 cm

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Subjects / Keywords:
Pharmaceutical chemistry -- Environmental aspects ( lcsh )
Water quality -- Analysis ( lcsh )
Solid-phase analysis ( lcsh )
Liquid chromatography ( lcsh )
Water -- Pollution -- Measurement ( lcsh )
Drugs -- Environmental aspects ( lcsh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Bibliography:
Includes bibliographical references (leaves 108-112).
General Note:
Department of Chemistry
Statement of Responsibility:
by Jeffery Dene Cahill.

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University of Colorado Denver
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Auraria Library
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45224207 ( OCLC )
ocm45224207
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LD1190.L46 2000m .C34 ( lcc )

Full Text
DETERMINATION OF HUMAN PHARMACEUTICALS IN SURFACE WATER
USING SOLID-PHASE EXTRACTION AND
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
by
Jeffery Dene Cahill
B.S., University of Colorado, 1997
A thesis submitted to the
University of Colorado at Denver
in partial fulfillment
of the requirements for the degree of
Master of Science
Chemistry
2000


This thesis for the Master of Science
degree by
Jeffery Dene Cahill
has been approved
by
Mark R. Burkhardt
zo .ZOO^>
Date


Cahill, Jeffery Dene (M.S., Chemistry)
Determination of Human Pharmaceuticals in Surface Water Using Solid-Phase Extraction
and High-Performance Liquid Chromatography-Mass Spectrometry.
Thesis directed by Professor Larry G. Anderson
ABSTRACT
A method for determining low pg/lto low ng/l concentrations of human
pharmaceutical compounds and metabolites in surface water using solid-phase extraction
and high-performance liquid chromatography-mass spectrometry has been developed. A list
of pharmaceutical compounds that were expected to be found in surface water was
developed using published market research and available dosing information. High-
performance liquid chromatography-mass spectrometry using positive ionization electrospray
interface and selected ion monitoring was used for qualitative and quantitative analysis of the
compounds. High-performance liquid chromatography was performed using a narrow bore
column containing a base deactivated octadecyl silane stationary phase. A 10mM
ammonium formate/formic acid buffer, pH 3.7, and acetonitrile organic solvent was used for
the mobile phase in stepped gradients. Concentrations of compounds were determined by
selected ion monitoring for increased sensitivity and quantitation software capable of
extracting the ions of choice for quantitation using an internal standard. Relative abundance
of fragment ions for each compound further verified compound identification. Solid-phase
extraction using N-vinyl pyrrolidone modified styrene divinylbenzene sorbent gave recoveries
greater than 60% for twelve of nineteen compounds tested and standard deviations less than
15%. Seventy-seven 1-liter field samples were collected from various regions across the
m


U.S. and shipped over-night for extraction and analysis. The presence of 12 of the 22
analytes tested for was determined in concentrations ranging from 9 parts per trillion to 10
parts per billion. Non-prescription compounds such as acetaminophen, caffeine, and the
nicotine metabolite cotinine were measured in the highest relative concentrations up to 10
pg/l. Compounds such as gemfibrozil, caffeine, ibuprofen, sulfamethoxazole, and
trimethoprim, previously detected in other studies were measured at median concentrations
between 0.300 and 0.550 pg/l. New compounds not previously reported such as
acetaminophen, cotinine, cimetidine, paraxanthine, diltiazem, and dehydronifedipine were
detected at median concentrations between 0.011 and 0.600 pg/l. Quality control set-spike
recoveries (n=8), performed with sample preparation and analysis for all compounds
detected, yielded relative standard deviations of less than 18%.
This abstract accurately represents the content of the candidates thesis. I recommend its
publication.
Signed
IV


DEDICATION
I dedicate this thesis to my parents, Gary and Nancy, my wife Caryn, and my children,
Chelsea, Mathew, and Conner for your support and inspiration in achieving this work.


ACKNOWLEDGEMENTS
Dr. Edward T. Furlong, US Geological Survey, NWQL, Dr. Mark R. Burkhardt, US Geological
Survey, NWQL, and Dr. Larry G. Anderson, University of Colorado, Denver for cooperative
support.
Herb Buxton, Toxic Substances Hydrology Program, US Geological Survey, provided
financial support.
Dana Kolpin, Iowa District, US Geological Survey, coordinated nationwide sample collection.
Dan Hippe, Georgia District, US Geological Survey, and Deborah Moll, Centers for Disease
Control and Prevention, provided support and samples.
Barbara Kemp and Jeff Stewart, National Water Quality Laboratory, MSW, SPE, and FID
support.


CONTENTS
Figures......................................................................... x
Tables.......................................................................... xiii
Chapter
1. Introduction.............................................................. 1
1.1 Pharmaceutical Compounds in the Environment............................... 1
1.2 Incomplete Removal of Drugs by Sewage Treatment Processes................. 3
1.3 Solid-Phase Extraction of Drug Compounds from Aqueous Media............... 3
1.3.1 Introduction.............................................................. 3
1.3.2 Solid-Phase Extraction of Drug Compounds.................................. 4
1.3.3 Chemistry of Drugs........................................................ 4
1.3.4 Sample Matrix............................................................. 4
1.3.5 Solid-Phase Adsorbent Material............................................ 5
1.4 High Performance Liquid Chromatography-Mass Spectrometry.................. 7
1.4.1 Introduction.............................................................. 7
1.4.2 High Performance Liquid Chromatography.................................... 7
1.4.3 Electrospray Interface for HPLC-MS........................................ 9
1.4.4 Mass Spectrometry........................................................ 10
2. Methods.................................................................. 11
2.1 Pharmaceutical Compounds................................................. 11
2.2 HPLC-MS.................................................................. 12
2.3 Solid-Phase Extraction................................................... 14
2.4 Sample Collection........................................................ 16
vii


2.5 Sample Preparation and Analysis: Final Method............................ 17
3. Results.................................................................. 18
3.1 Target Analyte Determination............................................. 18
3.2 High Performance Liquid Chromatography................................... 24
3.3 Mass Spectral Characterization of Compounds.............................. 26
3.4 Data Processing and Calibration Curves................................... 30
3.5 Solid-phase Extraction................................................... 31
3.5.1 Solid-phase sorbent trials............................................... 31
3.5.2 SPE Break-through........................................................ 63
3.6 Method Detection Level................................................... 63
3.7 Environmental Sample Analysis............................................ 64
4. Discussion.......................:...................................... 75
4.1 Target Analytes.......................................................... 75
4.2 High Performance Liquid Chromatography................................... 80
4.3 Mass Spectral Characterization of Compounds.............................. 80
4.4 Data Processing.......................................................... 89
4.5 Solid-phase Extraction................................................... 92
4.5.1 Carbopak Results......................................................... 92
4.5.2 HLB Results.............................................................. 93
4.5.3 ENV+ Results............................................................. 94
4.5.4 C-18 Results............................................................. 94
4.5.5 SPE Sorbent Selection.................................................... 96
4.6 Method Detection Level................................................... 98
4.7 Environmental Sample Analysis............................................ 99
5. Conclusion.............................................................. 106
viii


References
108
IX


FIGURES
Figure
1.1 Four classes of drug compounds based on ionic classification.................... 4
1.2 A. Polymeric Styrene divinylbenzene, and B. N-vinylpyrrolidone.................. 6
2.3.1 Solid-phase extraction apparatus............................................... 16
3.2.1 Chromatogram produced by initial linear gradient, column temp 40C............. 25
3.2.2 Chromatogram produced by final stepped gradient, column temp 27C.............. 26
3.3.1 Flow injection analysis of isotopic phenacetin, integrated TICs,
and corresponding mass spectra............................................... 27
3.3.2 TIC showing selected ion monitoring windows and fragmentor voltage setting.. 29
3.5.1.1 Average recovery, Carbopak SPE cartridges. (ACN/MeOH is 30:70; N/A
refers to neutral followed by acidic solvent.)............................... 55
3.5.1.2 Carbopak SPE cartridge average recovery adjusted for sample in ACN acid.... 56
3.5.1.3 Standard deviation, Carbopak SPE average recovery.............................. 56
3.5.1.4 Average recovery HLB SPE cartridges............................................ 57
3.5.1.5 Standard deviation, HLB SPE average recovery................................... 57
3.5.1.6 Average recovery, ENV+ SPE cartridges.......................................... 58
3.5.1.7 ENV+ SPE cartridge average recovery adjusted for sample in MeOH acid.......... 58
3.5.1.8 Standard deviation, ENV+ average recovery...................................... 59
3.5.1.9 Average recovery, C-18 SPE cartridges.......................................... 59
3.5.1.10 C-18 SPE cartridge average recovery adjusted for sample in ACN acid........... 60
3.5.1.11 Standard deviation, C-18 average recovery...................................... 60
3.5.1.12 Highest recovery for Carbopak SPE cartridges, acidic MeOH: acetonitrile....... 61
3.5.1.13 Highest recovery for C-18 SPE cartridges, acidic methanol/acetonitrile........ 62
x


3.5.1.14 Highest recovery for ENV+ SPE cartridges, acidic methanol/acetonitrile....... 62
3.5.1.15 Highest recovery for HLB SPE cartridges, methanol/acidic methanol............. 63
4.3.1 Metformin, formula wt. 129, fragments 113, 130, fragmentor 80V................ 81
4.3.2 Amoxicillin, formula wt. 365, fragments 114, (208 not interpreted), 349,
fragmentor 70V(Straub and Voyksner 1993)..................................... 81
4.3.3 Cotinine, formula wt. 176, fragments 177, 98, 80, fragmentor 90V.............. 81
4.3.4 Salbutamol, formula wt. 239, fragments 240, 222, 166, fragmentor 70V.......... 82
4.3.5 Cimetidine, formula wt. 252, fragments 253, 159, (117 not interpreted),
fragmentor 80 V.............................................................. 82
4.3.6 Acetaminophen, formula wt. 151, fragments 152, 110, 93, fragmentor 87V........ 82
4.3.7 Ranitidine, formula wt. 314, fragments 315, 270, (176 not interpreted),
fragmentor 80 V.............................................................. 83
4.3.8 1,7-Dimethylxanthine, formula wt. 180, fragments 181, 124, fragmentor 88V... 83
4.3.9 Lisinopril, formula wt. 405, fragments 406, 246............................... 83
4.3.10 Caffeine, formula wt. 194, fragments 195, 138, 110, fragmentor 100V........... 84
4.3.11 Enalaprilat, formula wt. 348, fragments 349, 303, 206, fragmentor 100V........ 84
4.3.12 Trimethoprim, formula wt. 290, fragments 291,261,230, fragmentor 120V......... 84
4.3.13 Digoxigenin, formula wt, 390, fragments 391,373, 355, fragmentor 100V......... 85
4.3.14 Paroxetine metabolite, formula wt 331, fragments 332, 192, 109,
fragmentor 120V.............................................................. 85
4.3.15 Diltiazem, formula wt. 414, fragments 415, 370, 178, fragmentor 105V.......... 85
4.3.16 Furosemide, formula wt. 330, fragments 81, 353, fragmentor 120V............... 86
4.3.17 Ibuprofen, formula wt. 207, fragments 161,207, fragmentor 60V................. 86
4.3.18 Warfarin, formula wt. 308, fragments 163, 251, 309, fragmentor 70V............ 86
4.3.19 Dehydronifedipine, formula wt. 344, fragments 268, 284, 345,
fragmentor 100V.............................................................. 87
4.3.20 Gemfibrozil, formula wt. 250, fragments 205, 233, 251, fragmentor 50V......... 87
xi


4.3.21 Sulfamethoxazole, formula wt. 253, fragments 156, 254, fragmentor 80V.. 87
4.3.22 Fluoxetine, formula wt. 309, fragments 148, 227, 310, fragmentor 70V... 88
4.3.23 HPLC-MS Ion transport optics........................................... 89
4.4.1 Caffeine 13C vs Caffeine response, ion suppression..................... 90
4.4.2 Calibration curve for acetaminophen. Levels 1-7........................ 91
4.4.3 Calibration curve for lisinopril. Levels 3-7........................... 91
4.5.4.1 ENV+Recovery, subsequent study............................... ........ 97
4.7.1 Chromatogram of STE.................................................... 99
4.7.2 TIC, sample no. 992420018, set no. 9285.06 (table 3.7.5).............. 100
xii


TABLES
Table
2.3.1 Solid-phase extraction solvent scheme for each cartridge type................. 15
3.1.1 Top 20 prescription drugs, high mass drugs, and non-prescription drugs.... 18
3.1.2 Drug compounds and metabolites in order of estimated mass prescribed...... 20
3.1.3 Target analytes for determination of pharmaceutical compounds in
surface water............................................................. 21
3.2.1 Pump timetable for initial chromatographic conditions......................... 25
3.2.2 Pump timetable for final chromatographic conditions........................... 26
3.3.1 Flow injection analysis results, optimum fragmentor voltage and major ions.... 27
3.3.2 Selected ion monitoring windows, fragmentor voltage and ion assignments.... 29
3.4.1 Calibration curve type and coefficient of regression for one calibration.. 30
3.5.1.1 Percent recovery, carbopak eluted with methanol.............................. 31
3.5.1.2 Percent recovery, carbopak eluted with methanol pH 3.7....................... 32
3.5.1.3 Percent recovery, carbopak eluted with methanol/methanol pH 3.7.............. 33
3.5.1.4 Percent recovery, carbopak eluted with methanoliacetonitrile (70:30)......... 34
3.5.1.5 Percent recovery, carbopak eluted with methanoLacetonitrile pH 3.7........... 35
3.5.1.6 Percent recovery carbopak, eluted with methanoLacetonitrile/methanol:
acetonitrile pH 3.7....................................................... 36
3.5.1.7 Percent recovery, HLB eluted with methanol................................... 37
3.5.1.8 Percent recovery, HLB eluted with methanol pH 3.7............................ 38
3.5.1.9 Percent recovery, HLB eluted with methanol/methanol pH 3.7................... 39
3.5.1.10 Percent recovery, HLB eluted with methanoLacetonitrile (70:30)............... 40


3.5.1.11 Percent recovery, HLB eluted with methanol:acetonitrile pH 3.7........... 41
3.5.1.12 Percent recovery HLB, eluted with methanohacetonitrile/
methanohacetonitrile pH 3............................................................ 42
3.5.1.13 Percent recovery, ENV+ eluted with methanol.............................. 43
3.5.1.14 Percent recovery, ENV+eluted with methanol pH 3.7....................... 44
3.5.1.15 Percent recovery, ENV+ eluted with methanol/methanol pH 3.7.............. 45
3.5.1.16 Percent recovery, ENV+ eluted with methanohacetonitrile (70:30........... 46
3.5.1.17 Percent recovery, ENV+ eluted with methanohacetonitrile pH 3.7........... 47
3.5.1.18 Percent recovery ENV+, eluted with methanobacetonitrile/methanol:
acetonitrile pH 3.7........................................................ 48
3.5.1.19 Percent recovery, C-18 eluted with methanol............................... 49
3.5.1.20 Percent recovery, C-18 eluted with methanol pH 3.7........................ 50
3.5.1.21 Percent recovery, C-18 eluted with methanol/methanol pH 3.7............... 51
3.5.1.22 Percent recovery, C-18 eluted with methanol:acetonitrile (70:30).......... 52
3.5.1.23 Percent recovery C-18, eluted with methanol:acetonitrile pH 3.7........... 53
3.5.1.24 Percent recovery C-18, eluted with methanoLacetonitrile/methanol:
acetonitrile pH 3.7........................................................ 54
3.6.1 MDL in ng/ml on column amount............................................ 64
3.7.1 Sample set number 10-5 results........................................... 66
3.7.2 Sample set number 9279.10 results...................................... 67
3.7.3 Sample set number 9279.11 results...................................... 68
3.7.4 Sample set number 9285.05 results...................................... 69
3.7.5 Sample set number 9285.06 results...................................... 70
3.7.6 Sample set number 9287.04 results...................................... 71
3.7.7 Sample set number 9287.06 results...................................... 72
3.7.8 Sample set number 9292.03 results...................................... 73
3.7.9 Sample result summary.................................................... 74
XIV


3.7.10 Spike recovery summary........................................................ 75
4.1.1 Determination of target analytes from biotransformation references............ 78
4.7.1 Summary of sample detections................................................. 101
4.7.2 Set-spike recovery summary................................................... 103
4.7.3 CCV summary, 0.10 pg/l concentration......................................... 105
xv


1. Introduction
1.1 Pharmaceutical Compounds in the Environment
Developing methodology for assessing water quality is one of the most relevant
pursuits in contemporary environmental analytical chemistry. In order to characterize water
quality, concentrations of inorganic and organic pollutants have to be accurately and
precisely determined, often at vanishingly low concentrations. Industrial chemicals,
herbicides, pesticides and their metabolites have been the major focus of water quality
analysis. Pharmaceutical compounds have only recently aroused interest in environmental
analysis (Hirsch, Ternes et al. 1998).
The use of drug compounds to cure and avert illness and disease is an integral part
of modern culture. Many tons of drug compounds for human and veterinary use are
produced each year (Table 3.1.1). The exact amounts of drug compounds produced are not
published in the literature but can be estimated by multiplying the daily dose with the number
of prescriptions and the average duration of a prescription in days (Ternes 1998).
Recent studies have revealed the presence of various pharmaceutical compounds at
very low concentrations in environmental water samples. These drug compounds persist to a
significant degree through wastewater treatment and are then introduced into surface water
in the environment (Stumpf, Ternes et al. 1999). The anticancer drug bleomycin was found in
sewage treatment effluents (STE) and river water samples in southeast England (Aherne,
Hardcastle et al. 1990). Such antineoplastic drugs have been reported to be carcinogenic,
mutagenic and teratogenic. Aherne and associates used radioimmunoassay techniques to
determine bleomycin levels of between 0.011 and 0.019 jj.g/1 in STE and < 0.005 and 0.017
(j.g/1 in river water, and drinking water supplies.
1


1.2 Incomplete Removal of Drugs by
Sewage Treatment Processes
Incomplete removal of drug compounds upon passage through STP has been
demonstrated in Brazilian and German STP. Concentrations of drugs in Brazilian STP
influents and corresponding effluents were measured and showed removal rates between 34
and 83% (Stumpf, Ternes et al. 1999). The drug ibuprofen had removal rates up to 75% via
activated sludge treatment while only 46% of gemfibrozil, an antihyperlipidemic, were
removed. A similar sampling scheme was used in a study of a German STP and likewise
showed a general removal of 60% of the load of drug residues detected in the influent
(Ternes 1998). It should be noted that there are no known investigations comparing
adsorption to sewage solids versus degradation of drug compounds during passage through
STP as the mechanism of removal.
1.3 Solid-Phase Extraction of Drug Compounds
from Aqueous Media
1.3.1 Introduction
Solid-phase extraction has become established as a very effective method for
sample preparation (Theodoridis, Papadoyannis et al. 1995). SPE is a method of sample
preparation for concentrating and purifying analytes from a solution by sorption onto a solid-
phase material. The most common modes of sorption include normal phase, reversed
phase, and ion exchange. Analytes are loaded onto the sorbent material by passing through
in the liquid matrix and partitioning into the sorbent. They are then eluted with a solvent
amenable to the instrumental analysis being performed. The analytes will partition from the
solid phase packing into the solvent. The practical advantages offered by SPE over liquid-
liquid extraction techniques include higher efficiency, small quantities of organic solvents
being used, fewer emulsion problems, ease of handling, and amenability to automation
(Theodoridis, Papadoyannis et al. 1995; Thurman and Mills 1998).
3


1.3.2 Solid-Phase Extraction of Drug Compounds
SPE is widely used for sample preparation of forensic and clinical samples prior to
determination of pharmaceutical or drug residues (Theodoridis, Papadoyannis et al. 1995).
Among considerations for effective SPE of drug compounds are the chemistry of the drug,
(whether it is acidic, basic, or neutral), the matrix from which the drug is being extracted, and
the sorbent material to be used. (Thurman and Mills 1998).
1.3.3 Chemistry of Drugs
Drugs are classified chemically as acidic, basic, neutral, and amphoteric based on
the ionic state of the compound in solution at various pH values (Thurman and Mills 1998).
A basic compound contains a basic functional group such as an amine and accepts a proton
at low pH becoming a cation. An acidic compound contains an acidic group such as a
carboxylic acid which donates a proton at pHs generally above 5 becoming anionic. Drugs
that contain neither acidic nor basic functional groups are neutral. Amphoteric compounds
contain both acidic and basic functional groups and may be cationic, anionic, or zwitterionic.
Figure 1.1 shows examples of each of the classes of drugs previously described.
(-)Cotinine
(Basic)
Warfarin
(Neutral)
Figure 1.1. Four classes of drug compounds based on ionic classification.
1.3.4 Sample Matrix
Sample matrices encountered in analysis of drug compounds typically are
4


bodily fluids such as plasma, serum, and urine. SPE of drug compounds from urine provides
some guidelines for environmental water SPE methodologies. Pretreatment of urine includes
filtration or centrifugation to remove particulate matter and also pH adjustment to neutralize or
acidify from a slightly alkaline pH of 7.5 (Thurman and Mills 1998). Sample pretreatment for
surface or ground water requires filtering prior to SPE and in some cases pH adjustment
depending on the nature of the compound being extracted.
1.3.5 Solid-Phase Adsorbent Material
Commonly used solid-phase sorbent materials for organic sample preparation
include graphitized carbon, bonded silica gels (hydrocarbon chains bonded through silyl ether
linkages), and polymeric resins (Liu and Gadzala 1997). Mechanisms for adsorption to SPE
materials include van der Waals attraction and n bond electron donating for nonpolar
interaction, hydrogen bonding for polar interaction, and interactions of ions of opposite charge
for ion-exchange (Liu and Gadzala 1997; Thurman and Mills 1998).
Graphitized carbon sorbents are polymeric and offer a high surface area for
increased capacity. They act through hydrophobic interaction and have the potential to act
with polar molecules via suspected carbon-oxygen complexes, thus behaving as a mixed
mode sorbent (Thurman and Mills 1998). A negative aspect of graphitized carbon sorbents is
irreversible bonding of analytes to the sorbent materials causing retention of analytes even in
the presence of organic solvents.
Bonded silicas are among the most commonly used solid phases for the extraction of
drugs of abuse from urine specimens (Liu and Gadzala 1997). The most common bonded
phase is the C-18 phase. The C-18 phase is used widely for extraction from aqueous
matrices and for a wide range of analyte polarities. Bonded silicas have much lower surface
area than polymeric sorbents by weight, and cannot operate over extreme values of pH
(Thurman and Mills 1998).
5


Another polymeric sorbent is styrene-divinylbenzene (SDB). The typical surface area
of SDB is 600-1200 m2/g as compared to 550 m2/g for the average pore size of bonded silica
(Thurman and Mills 1998). Recent improvements in the manufacture of polymeric sorbents
have minimized the occurrence of interferences resulting from impurities leaching from the
sorbents. In addition to greater surface area the aromatic rings of the sorbent permit electron
donor interactions between the n bonds of the sorbent and the solute, further enhancing
extraction capacity. Some manufacturers add polar groups to the SDB matrix to enhance
water movement through the sorbent, and to enhance mass transfer. International Sorbent
Technologies sulfonates its SDB and Waters Corp. adds N-vinylpyrrolidone for these
purposes (Thurman and Mills 1998). The structures of SDB and N-vinylpyrrolidone are
represented in figure 1.2 and it is assumed that the N-vinylpyrrolidone incorporates into the
polymer in the same manner, as does styrene.
Figure 1.2. A. Polymeric styrene divinylbenzene, and B. N-vinylpyrrolidone.
6


1.4 High Performance Liquid Chromatography-
Mass Spectrometry
1.4.1 Introduction
Valid determinations for concentrations of compounds in any matrix require analytical
methods that are highly accurate and reliable. Combinations of mass spectrometry with
suitable chromatographic procedures are the methods of choice because of sensitivity,
specificity, and precision (Maurer 1998). Pharmaceutical compounds are generally polar and
thermally labile making them better suited for chromatographic separation via HPLC without
the need for derivatization called for by gas chromatography (Castillo, Alpendurada et al.
1997). Traditionally, analysis of pharmaceuticals has been performed using HPLC with UV
detection. For the simultaneous analysis of a variety of compounds it is difficult to obtain
adequate separation for analysis using UV detection. UV absorbing non-target compounds
coextracted with the analytes of interest also interfere with absorbance spectra. To
overcome this obstacle mass spectrometric detection with selected ion monitoring can be
used to obtain specific and quantitatable data when compounds arent sufficiently separated
for analysis via elution peak area as determined by UV detection. Therefore, HPLC coupled
with mass spectrometry is an ideal means for simultaneous determination of concentrations
of a variety of pharmaceutical compounds.
1.4.2 High Performance Liquid Chromatography
1.4.2.1 Column Packing
Successful chromatography requires the optimum balance of intermolecular forces
between the mobile phase, the solute, and the stationary phase. More than three fourths of
all HPLC separations are performed with reversed-phase, bonded octyl or octadecyl siloxane
packing (Skoog, West et al. 1992). Ionic solutes, especially basic compounds, can interact
with the silanols of silica-based columns leading to increased retention, band tailing, and
7


column to column irreproducibility (Snyder, Kirkland et al. 1997). Selecting a column that is
designed for use with basic compounds can reduce silanol interactions. The packing in these
columns when fully hydroxylated favors the highest concentration of geminal and associated
silanols over free silanols which are highly acidic and reactive with basic compounds. Silanol
effects can also be reduced by using a significant buffer concentration (>10mM), and
choosing buffer cations that are strongly retained by the silanols such as sodium, potassium,
ammonium, triethylammonium, or dimethyloctylammonium (Snyder, Kirkland etal. 1997).
1.4.2.2 Column Dimension
Narrow-bore columns of 0.21 cm characteristically produce a fourfold decrease in
solvent use as compared to the widely used 0.46-cm units. The major advantage of narrow-
bore columns is higher sample mass detection sensitivity. This is especially useful when
interfacing HPLC with mass spectrometers that require solvent input volumes as low as 0.200
ml/min (Snyder, Kirkland et al. 1997).
1.4.2.3 Mobile Phase
Buffer solubility, stability, viscosity, temperature, interactions with the solute, column
and equipment, and volatility are considerations in HPLC/MS method development. It is
useful to know the approximate pKa of sample compounds when optimizing mobile phase pH.
Aliphatic amines have pKa values in the range of 8-10, while aromatic amines, pyridines, and
aromatic and aliphatic carboxylic acids are on the low end with pKa values of 4-5 (Snyder,
Kirkland et al. 1997). The previously mentioned functional groups all are present in drug
compounds therefore an effective buffer would maintain pH at just below 4 to ensure neutral
and cationic solutes for positive electrospray HPLC/MS interfacing.
Volatile buffers are important when HPLC is interfaced with mass spectrometry to
enhance droplet evaporation and formation of gas-phase ions for detection. Buffers such as
8


ammonium carbonate, ammonium formate, ammonium acetate, and trifluoroacetic acid are
considered effective for this purpose (Snyder, Kirkland et al. 1997).
Solvent type affects selectivity for ionic compounds in the same manner as for neutral
compounds. Selection of solvent depends on requirements for separation of sample
compounds and solubility characteristics with the mobile-phase buffer. While methanol is
more polar and usually more selective for polar compounds, acetonitrile exhibits increased
mobile-phase strength at lower concentration and is more selective with lower polarity
compounds (Snyder, Kirkland et al. 1997).
1.4.3 Electrospray Interface for HPLC-MS
To make mass analysis possible, HPLC-MS interfaces must convert analyte from
liquid to gas phase, separate and remove solvent from analyte, reduce sample at
atmospheric pressure to low pressure, and ionize sample analyte (Willoughby, Sheehan et al.
1998). Over the past three decades different LC-MS interfaces including moving belt, fast
atom bombardment, thermospray, and atmospheric pressure ionization including chemical
ionization (APCI), and electrospray ionization (ESI) have been developed to remove the
analyte from the mobile phase and ionize it for mass analysis (Maurer 1998). In terms of the
molecular mass range and increased polarity of analytes, ESI is the most universal among
the interfacing techniques previously mentioned (Maurer 1998; Willoughby, Sheehan et al.
1998).
ESI allows the determination of high mass molecules up to several hundred
kilodaltons and/or very polar molecules such as quaternary amines, sulfate conjugates,
nucleotides, and phospholipids (Maurer 1998). A prerequisite for gas phase ion production
with ESI is that the analyte is ionized in solution so that virtually any ion in solution can be
observed with the mass spectrometer (Willoughby, Sheehan et al. 1998). ESI is a soft
ionization technique, which produces an abundance of MH ions, and/or adduct ions. In
9


addition, collisional fragmentation of analyte molecules can be induced in the ion source or by
tandem mass analysis (MS-MS) to enhance molecular identification (Maurer 1998).
1.4.4 Mass Spectrometry
Mass spectrometry coupled with the appropriate method for separation is an
analytical method of choice for qualitative and quantitative determinations (Maurer 1998).
For quantitative analysis, the more ion currents of specific mass-charge ratios (m/z) that are
monitored in a given mass spectrum the higher the confidence that a compound of interest is
present in the sample. Conversely, monitoring a large number of ion currents leads to a
diminished statistical representation of any given ion current. Selected ion monitoring (SIM)
refers to the dedicated use of a mass spectrometer to acquire and record ion current at only
selected m/z values, in contrast to scan mode which acquires ion currents for all m/z values
in a range. High sensitivity can be achieved if a mass spectrometer is acquiring the ion
current at only a few m/z values, approximately 1000 times more sensitive than the full
scanning mode (Watson 1997). The quadrupole mass analyzer is well suited to SIM because
any region of the mass spectrum can be monitored without altering optimum conditions in the
ion source or the mass analyzer, and the mass axis can be traversed rapidly with good
response and no drift (Watson 1997).
10


2. Methods
2.1 Pharmaceutical Compounds
Identification of compounds of interest was performed using printed and electronic
reference material indicating the most widely prescribed drugs and available dosing
information (RxList; Maurer 1998; Ternes 1998). Pharmaceutical compounds were selected
for the most part by the greatest mass prescribed as determined by multiplying the number of
prescriptions by the unit dose and the number of units per prescription, equation 2.1.1
(Ternes 1998). Major metabolites or eliminants of compounds per literature review were
selected if available. In addition, a number of non-prescription drugs are assumed to be
consumed in large masses and were also included in the study. Some compounds that had
very low mass per unit but high numbers of prescriptions were selected as an exploratory
facet of the research.
(Number of prescriptions) x (Unit dose) x (Units per prescription)=Mass Prescribed Eq.2.1.1
The following compounds were purchased from Sigma: Acetaminophen, 3-(a-
Acetonylbenzyl)-4-hydroxycoumarin, Amoxicillin, Aspirin, (-)-Cotinine, Digoxigenin, Digoxin,
Diltiazem Hydrochloride, 1,1-Dimethylbiguanide Hydrochloride, 1,7-Dimethylxanthine,
Enalapril Maleate, Furosemide, Gemfibrozil, Ibuprofen, Lisinopril, Salbutamol,
Sulfamethoxazole, Trimethoprim.
The compounds Cimetidine, Fluoxetine, Ranitidine, and Theophylline were
purchased from RBI. Phenacetin-ethoxy-1-13C, and Caffeine-3-methyl-13C were purchased
from Cambridge Isotope Laboratories.
11


The following compounds were generously donated by their respective
manufacturers: Dehydronifedipine, Bayer AG; Enalaprilat, Merck & Co., Inc.; Paroxetine, and
Paroxetine Related Compound (BRL36610), SmithKIine Beecham Pharmaceuticals.
Stock solutions of all compounds, spiking solutions, and surrogate solutions were in
methanol, Burdick & Jackson, High Purity Solvent. Calibration solutions were prepared using
stock solutions then brought to volume in formate buffer.
2.2 HPLC-MS
Ammonium formate/formic acid buffer (10mM, pH 3.7) for HPLC was prepared from
1M solutions of ammonium formate and formic acid, 40ml and 48ml respectively, diluted to
4000ml in Burdick & Jackson, high purity solvent grade water. The 1M solution of ammonium
formate consisted of 65.69g formic acid, ammonium salt, JT Baker, minimum 96% purity in 1
liter of Burdick & Jackson, high purity solvent grade water. The 1M formic acid solution
consisted of 38.8ml formic acid, EM Science, minimum 98% purity in 1 liter of Burdick &
Jackson, high purity solvent grade water. The organic solvent used was Acetonitrile UV,
Burdick & Jackson, high purity solvent. The flow rate for HPLC was 0.200 ml/min. A linear
gradient, 20 to 90% organic was used in initial tests of single compounds (see table 3.2.1,
and figure 3.2.1). A stepped gradient was used to achieve chromatography for the mix of
compounds: 6% organic for the first 5 minutes then increased to 14% at nine minutes, 24% at
10 minutes, 41% at 15 minutes, 51% at 16 minutes 70% at 26 minutes, and 100% at 27
minutes (see table 3.2.2, and figure 3.2.2).
The analytical column used was a MetaChem, Metasil Basic 3pm, 150 x 2.0 mm,
ODS column. Guard columns used were a MetaChem, Metasil Basic Safeguard, 3pm, 2.0
mm guard column, and a NewGuard RP-18, 7pm, 15 x 3.2 mm.
Instrumentation consists of a Hewlett Packard 1100 HPLC system including vacuum
degasser, autosampler, thermostatted column compartment, and UV-Vis diode array
12


detector. The mass spectrometer is a Hewlett Packard 1100 MSD using an atmospheric
pressure ionization/electrospray interface with nitrogen for the nebulizing gas. Spray
chamber settings are nebulizer pressure of 20-psi, 9 l/min drying gas flow, and drying gas
temperature of 350 C. Full scan analysis was performed over the range of 50-850 m/z at a
scan rate of 2.03 seconds per scan.
Target ions for selected ion monitoring were determined by flow-injection analysis
whereby the analytical column is bypassed and a 5 ul sample of a 1ppm standard is injected
into the MSD to determine optimum fragmentation. Peak areas and fragmentation
characteristics were compared for a series of injections made at varying fragmentor voltages
beginning at 50, 60, 70, 80, and 90V then either higher or lower if lack of or excessive
fragmentation necessitated.
Data processing for quantitation is accomplished using Thru-Put Systems Inc., Target
Chromatographic Analysis Software, Target/NT Revision 4.00. Up to seven calibration levels
including 0.0100, 0.0500, 0.100, 0.200, 0.400, 0.800, and 2.00 ng/pl on-column are used for
each compound, and a calibration set was included with each sample set. Quantitation is
based on an internal standard using Caffeine-3-methyl-13C at 1 .OOng/pl, and calibration
curves used are either linear or quadratic based upon best fit and coefficient of regression
greater than 0.995. The predominant ion in the mass spectra of each compound with the
exception of caffeine is used as the quantitative ion for that compound. One or preferably two
other characteristic ions, optimally greater than 10% of the abundance of the quantitative ion,
are used as qualifiers in comparing relative abundance for identification. For sample
concentration computations the on-column amount as determined by peak area compared to
internal standard is multiplied by a dilution factor of one-thousand and divided by the sample
volume extracted (Equation 2.2.1).
13


(Amount on-column) x (Dilution factor) x 1/(Volume extracted) = Concentration Eq. 2.2.1
The method detection level was determined according to U.S. Environmental
Protection Agency guidelines (EPA 1992). The MDL is defined as the minimum
concentration of a substance that can be measured and reported with a 99% confidence that
the analyte concentration is greater than zero and is determined from analysis of a sample in
a given matrix containing the analyte. The MDL was determined by spiking eight samples of
pristine water taken from a mountain stream above known septic facilities and processing
each through the entire method. The standard deviation of the replicate measurements
(Equation 2.2.2) is calculated and the value is multiplied by the Students t value for (n-1)
degrees of freedom (Equation 2.2.3).
2.3 Solid-Phase Extraction
In order to determine the most appropriate SPE sorbent, solid-phase extraction trials
were performed using Waters, Oasis HLB, 0.5g, 6ml cartridges; International Sorbent
Technologies, Isolute, EC C-18, and ENV+, 0.5g, 6ml cartridges; and Supelco,
Supelclean ENVI-Carb, 0.5g, 6ml cartridges. Oasis HLB cartridges were selected and
used for extraction of environmental samples.
Eq. 2.2.2
Eq. 2.2.3
14


Each trial consisted of loading 1000 ng spiked samples in approximately 950 ml of
purified water (Solution 2000 Reagent Grade Water Purification Systems) onto selected
cartridges using a positive pressure FMI reciprocating pump at a rate of 15 ml per minute.
The four types of cartridges were tested using four different methods of elution each with
different solvents as represented in table 2.3.1.
The conditions for extraction and elution were as follows. SPE cartridges were
conditioned with 6 ml of methanol followed by vacuum drying at 12mmHg on a vacuum
manifold for 10 to 15 seconds. The cartridges were then conditioned with 6 ml of reagent
grade water and placed on the pumping apparatus. Samples contained in one-liter amber
glass bottles were placed with the pumping apparatus and tubing inserted into the bottle.
The FMI pumps were set to a rate of 15 ml per minute and the sample pumped through the
extraction cartridge and the pump turned off immediately after complete removal of the water
sample (figure 2.3.1). The cartridges were then washed to reduce highly polar interferences
by pulling 1 ml of 5% methanol in reagent grade water through the cartridge over a vacuum
manifold. For the breakthrough test, SPE cartridges were piggybacked and extraction and
elution proceeded.
Table 2.3.1. Solid-phase extraction solvent scheme for each cartridge type.
MeOH MeOH pH 3.7 MeOH/ MeOH pH 3.7 ACN:MeOH 70:30 ACN:MeOH pH 3.7 ACN:MeOH/ ACN:MeOH pH 3.7
Sample 1 Sample 1 Sample 1 Sample 1 Sample 1 Sample 1
Sample 2 Sample 2 Sample 2 Sample 2 Sample 2 Sample 2
Sample 3 Sample 3 Sample 3 Sample 3 Sample 3 Sample 3
Breakthrough Breakthrough
15


Fig. 2.3.1. Solid-phase extraction apparatus.
Elution of the analytes from the cartridges occurred according to the following
scheme. Trials for methanol and acetonitrile:methanol, 70:30, and each solvent acidified to
pH 3.7 with trifluoroacetic acid, Pierce, sequanal grade, were performed by eluting each
cartridge with three 3-ml aliquots of the solvent. Trials for the combination of neutral followed
by acidic solvent were carried out by first eluting with two 3 ml aliquots of neutral solvent
followed by two 2 ml aliquots of the acidified solvent.
The eluent was evaporated to dryness using a Zymark, TurboVap apparatus under
nitrogen at a pressure of 5 psi in a 40C bath for the initial SPE trials. For actual sample
preparation the eluent is evaporated to approximately 100liI. The sample is reconstituted
with 900|il of formate buffer (800pl for real samples) and 10Optl of isotopic caffeine internal
standard, 10ng/pl. Before vialing, the reconstituted sample was filtered through a Gelman
Laboratory, Acrodisc 13CR PTFE 0.2 pm, syringe filter.
2.4 Sample Collection
Environmental sample collection was performed according to National Water Quality
Assessment Program protocol (Shelton 1994) at various sites across the United States.
Surface water samples were collected in cleaned and burned amber glass bottles and were
shipped in coolers overnight to the testing facility. Samples were either filtered according to
16


protocol in the field, or are filtered upon receipt through 7pm glass fiber filters via positive
pressure reciprocating pump.
2.5 Sample Preparation and Analysis:
Final Method
A performance surrogate containing 1000ng of phenacetin-1-ethoxy-13C in 100pl of
methanol was added to each filtered sample. Each sample set consisted of no more than ten
samples, a blank containing performance surrogate, and a set spike with a concentration of
1.0 pg/l of all analytes in reagent water. The samples were extracted into Oasis HLB
cartridges at a rate of 15 ml/min. The cartridges were eluted with two 3-ml aliquots of
methanol followed by two 2-ml aliquots of acidified methanol. The eluent was gently vortexed
and then concentrated to approximately 100 pi under nitrogen in a 40C bath. The
concentrate was reconstituted with 100pl of caffeine-3-methyl-13C internal standard and 800
pi of the ammonium formate buffer. The extract was filtered through a 0.2 pm syringe filter
into a 2ml amber glass vial and capped.
A new calibration using fresh calibration samples was made for each HPLC/MS run.
Continuing calibration verification (CCV) samples were included in the instrument runs before
and after calibration sets and sample sets to verify calibration accuracy and precision. Data
files for each instrument run were transferred into a batch file and a data analysis method
was created for each batch based on the calibration. The data was analyzed and processed
using Target software.
17


3.
Results
3.1 Target Analyte Determination
Information for the most frequently prescribed drugs for the year 1998 was obtained
in the literature from compilation by commercial market tracking sources (Zoeller 1999). The
source of the prescription data was an audit that tracked 2.486 billion prescriptions dispensed
by 35,000 community pharmacies from December 1997 through November 1998, and
projected to provide national estimates. The top twenty prescription drugs were focused on
as well as non-prescription drugs commonly known to be frequently used (Zoeller 1999). In
addition, drugs that are high dose but lower in numbers of prescriptions were also selected
for the study. Table 3.1.1 contains the top twenty drugs as well as non-prescription drugs of
interest, and high mass drugs.
The compounds listed in table 3.1.1 were ordered according to total mass and some
compounds were eliminated based upon low mass or structural unsuitability (i.e. highly
lipophillic) for the experimental conditions. The compounds are reorganized according to
total mass and listed in table 3.1.2 together with the form of the compound, either metabolite
or parent compound, that is the predominant form that is excreted.
Table 3.1.1. Top 20 prescription drugs, high mass drugs, and non-prescription drugs.
Rank Drug name Generic name Classification Total Rxs x1000 Dosage mg* Units per Rx Total Mass kg
1 Premarin Tabs Conjugated estrogens Estrogens 41,316 0.625 21 542
2 Synthroid Levothyroxin Thyroid 34,709 0.1 30 104
3 Trimox Amoxicillin Antibacterial 31,281 500 27 422294
4 Hydrocodone/ APAP Hydrocodone/ APAP Opioid analgesic 30,747 7.5 20 4612
5 Prozac Fluoxetine Antidepressant 23,835 20 30 14301
6 Prilosec Omeprazole Anti-ulcer 23,586 20 30 14152
7 Zithromax Azithromycin Antibacterial 22,965 250 6 34448
18


Table 3.1.1 cont.
Rank Drug name Generic name Classification Total Rxs x1000 Dosage mg* Units per Rx Total Mass kg
8 Lipitor Atorvastatin Antihyperlipidemic 21,575 10 30 6473
9 Norvasc Amlodipine Antianginal, antihypertensive 20,838 5 30 3126
10 Claritin Loratadine Antihistamine 20,031 10 20 4006
11 Lanoxin Digoxin Cardiac glycoside 20,029 1 30 601
12 Zoloft Sertraline Antidepressant 19,183 50 30 28775
13 Albuterol Aerosol Salbutamol Antiasthmatic 18,272 6.7 1 122
14 Paxil Paroxetine Antidepressant 17,574 30 30 15817
15 Amoxicillin Amoxicillin Antibacterial 17,432 500 30 261480
16 Prempro Conjugated estrogens Estrogens 16,913 0.625 28 296
17 Zestril Lisinopril Antihypertensive 16,524 20 30 9914
18 Vasotec Enalapril Antihypertensive 16,340 10 30 4902
19 Augmentin Amoxicillin/ clavulanate Antibacterial 16,285 250 20 81425
20 Cephalexin Cephalexin Antibacterial 16,271 250 30 122033
22 Glucophage Metformin Antihyperglycemic 15,726 850 60 802026
23 Coumadin Tabs Warfarin Anticoagulant 14,653 2 20 586
26 Furosemide Oral Furosemide Diuretic 13,099 80 30 31438
28 Trimethoprim/ Sulfa Trimethoprim Antibacterial 12,317 80 20 19707
28 Trimethoprim/ Sulfa Sulfamethoxazole Antibacterial 12,317 400 20 98536
29 Cardizem CD Diltiazem Antiangina, antihypertensive 12,235 240 30 88092
34 Procardia XL Nifedipine Antianginal 10,971 60 30 19748
123 Zantac Ranitidine Histamine H2 inhibitor 4,078 150 60 36702
167 Gemfibrozil Gemfibrozil Antihyperlipidemic 2,785 600 60 100260
182 Tagamet Cimetidine Acetaminophen** Aspirin** Caffeine** Ibuprofen** Nicotine** Histamine H2 inhibitor Analgesic Analgesic CNS stimulant Analgesic, NSAID CNS stimulant 2,608 800 60 125184
* Dosages often vary, most usual dosage listed.
** No information to estimate mass available.
19


Table 3.1.2. Drug compounds and metabolites in order of estimated mass prescribed.
Rank Drug name Predominant Form Excreted Total Rxs x1000 Dosage mg* Units/Rx Total Mass kg
1 Trimox Amoxicillin 31,281 500 27 422294
Amoxicillin 17,432 500 30 261480
2 Glucophage Metformin 15,726 850 60 802026
3 Tagamet Cimetidine 2,608 800 60 125184
4 Gemfibrozil Hydroxymethyl/ Carboxyl 2,785 600 60 100260
5 Trimethoprim/Sulfa N4 Acetylated 12,317 400 20 98536
6 Cardizem CD Demethylated/ deacetylated 12,235 240 30 88092
7 Zantac Ranitidine 4,078 150 60 36702
8 Furosemide Oral Furosemide 13,099 80 30 31438
9 Zoloft Norsertraline 19,183 50 30 28775
10 Procardia XL Dehydronifedipine 10,971 60 30 19748
11 T rimethoprim/Sulfa Trimethoprim 12,317 80 20 19707
12 Paxil BRL 36610* 17,574 30 30 15817
13 Prozac Norfluoxetine 23,835 20 30 14301
14 Prilosec Hydroxyomeprazole 23,586 20 30 14152
15 Zestril Lisinopril 16,524 20 30 9914
16 Vasotec Enalaprilat 16,340 10 30 4902
17 Hydrocodone/ APAP Glucuronide/ Hydromorphone 30,747 7.5 20 4612
18 Lanoxin Digoxigenin 20,029 1 30 601
19 Coumadin Tabs 8-Hydroxywarfarin, 6-hydroxywarfarin 14,653 2 20 586
20 Albuterol Aerosol Salbutamol sulfate 18,272 6.7 1 122
NR* Acetaminophen** Glucuronide/sulfate 1000
NR* Aspirin** Hydroxyhippuric acid 650
NR* Caffeine** Paraxanthine 100
NR* Ibuprofen** Carboxyl, hydroxyl 400
NR* Nicotine** (-)-cotinine '20
* (-)trans-4-(4-fluoro-phenyl)-3-(4-hydroxy-3-
methoxyphenoxymethyl)piperidine
** Not rated
The compounds listed in table 3.1.2 were either purchased or requested from the
manufacturer. The compounds not obtained were norsertraline, hydroxyomeprazole, and
20


norfluoxetine. The compound hydroxyhippuric acid was studied but not included in the final
target analyte list. The final list of 22 target analytes is presented in table 3.1.3. The
metabolite forms of gemfibrozil, sulfamethoxazole, diltiazem, fluoxetine, warfarin, salbutamol,
acetaminophen and ibuprofen were not used and the parent compound was substituted.
Table 3.1.3. Target analytes for determination of pharmaceutical compounds in surface
water.
Common drug name Target analyte CAS No. (Mol. Wt.) Structure
Trimox, Amoxicillin Amoxicillin 61336-70-7 (365) HO OH
Glucophage Metformin 657-24-9 (129) NH NH 11 11 1 H
Tagamet Cimetidine 51481-61-9 (252)
Gemfibrozil Gemfibrozil 25812-30-0 (250) X o f i
Trimethoprim/Sulfa Sulfamethoxazole 723-46-6 (253) 0 '~-N
Cardizem CD Diltiazem 42399-41-7


Table 3.1.3 cont.
Common drug Target analyte CAS No. Structure
name
Mol. Wt.
Zantac Ranitidine 66357-35-5 YTT (314) ' r V / 0"
Furosemide Oral Furosemide 54-31-9 (330) !T HO Cl -/=0 y^rNH! 0
Procardia XL Dehydronifedipine 21829-25-4* ^ (Nifedipine) (344) oj N .S' / A. O 0
T rimethoprim/Sulfa Trimethoprim 738-70-5 \0 (290) /0"T| N NHj x f II nh2
Paxil BRL 36610* 61869-08-7* (Paroxetine) J. (331) X OMe
Prozac Fluoxetine 54910-89-3 F (309) F>S^ 1.^ H
Zestril Lisinopril 83915-83-7 (405) JZ o Y^ 0
22


Table 3.1.3 cont.
Common drug name Target analyte CAS No. Mol. Wt. Structure
Vasotec Enalaprilat 76420-72-9 (348) HO c%- jK'- OH
Lanoxin Digoxigenin 1672-46-4 (390) ho-
Coumadin Tabs Warfarin 81-81-2 (308)
Albuterol Aerosol Salbutamol 18559-94-9 (239) ' ' OH
Acetaminophen Acetaminophen 103-90-2 (151)
Caffeine Caffeine 58-08-2 (194)
Caffeine Paraxanthine 611-59-6 (180) XX>
23


Table 3.1.3 cont.
Common drug name Target analyte CAS No. Mol. Wt. Structure
Ibuprofen Ibuprofen 15687-27-1
(206) v ^^ ^0H
Nicotine (-)-cotinine 486-56-6 (176)
CAS number given is for parent compound.
3.2 High Performance Liquid Chromatography
HPLC of target compounds was developed starting with a linear gradient and
separation was optimized using stepped gradients in organic concentration in the mobile
phase. The initial gradient started with a two minute isocratic hold at 20% organic then
linearly increased to 90% over 40 minutes at a flow rate of 0.200 ml/min and a column
temperature of 40C (Table 3.2.1, Figure 3.2.1). The final optimum separation (Figure 2.2.2)
was achieved after numerous iterations of stepped gradient, as well as changes in flow rate
and column temperature. The final flow rate for optimum separation was 0.200 ml/min., and
the column temperature was 27C.


Table 3.2.1. Pump timetable for initial chromatographic conditions.
Time (min) %B (ACN) Flow (ml/min)
0 20 0.200
40 90
42 100
47 100
52 20
57 20
Figure 3.2.1. Chromatogram produced by initial linear gradient, column temp 40C, 1 ppb,
full scan.
25


Table 3.2.2. Pump timetable for final chromatographic conditions.
Time (min) %B (ACN) Flow (ml/min)
0 6 0.200
5 6
9 14
10 24
15 41
16 51
26 70
27 100
34 100
39 6
50 6
Figure 3.2.2. Chromatogram produced by final stepped gradient, column temp 27C, 0.1 ppb,
SIM.
3.3 Mass Spectral Characterization of Compounds
Mass spectra of the compounds were obtained by flow injection analysis. Peak
areas of compounds analyzed at varying fragmentor voltage settings were compared for
maximum intensity and relative abundance of ions (Figure 3.3.1). Optimum fragmentation
occurred when three distinctive ions formed with relative abundance greater than 10% for the
26


least abundant ion. Table 3.3.1 lists the optimum fragmentor voltage and most abundant ions
detected for each compound.
Figure 3.3.1 Flow injection analysis of isotopic phenacetin, integrated TICs, and
corresponding mass spectra.
Table 3.3.1 Flow injection analysis results, optimum fragmentor voltage and major ions.
Compound Fragmentor Voltage Fragments (m/z) Relative Abundance(%)
Metformin HCI 80 113,130* 15,100
Amoxicillin 90 114,208,349 100,15,5
Cotinine 90 80,98,177* 21,12,100
Salbutamol 70 166,222,240* 55,70,100
Cimetidine 80 117,159,253* 60,85,100
Acetaminophen 87 93,110,152* 14,100,78
Ranitidine HCI 80 176,270,315* 35,32,100
1,7,-dimethylxanthine 88 124,181* 52,100
27


Table 3.3.1 cont.
Compound Fragmentor Voltage Fragments (m/z) Relative Abundance (%)
Lisinopril 90 246,406* 15,100
Caffeine 100 110,138,195* 14,75,100
Enalaprilat 100 206,303,349* 100,20,40
Trimethoprim 120 230,261,291* 40,40,100
Digoxigenin 100 355,373,391* 100,36,15
Paroxetine metabolite 120 109,192,332* 14,45,100
Diltiazem HCI 105 178,370,415* 100,13,56
Furosemide 120 81,353** 25,100
Ibuprofen 60 161,207* 100,25
Warfarin 70 163,251,309* 60,20,100
Dehydronifedipine 120 268,284,345* 30,60,100
Gemfibrozil 50 205,233,251* 50,100,40
Phenacetin13C 90 110,139,181* 39,35,100
Sulfamethoxazole 80 156,254* 50,100
Fluoxetine HCI 70 148,227,310* 40,10,100
Caffeine-3-methyl-13C 100 111,139,198* 25,92,100
* (M+H)*
** Sodium adduct
Parameters for selected ion monitoring were set according to chromatographic separation
and effective fragmentor voltage. For co-eluting compounds, fragmentor voltage was set to
the lowest common effective voltage. SIM windows are shown in figure 3.3.2. For windows
that contained more than two compounds the number of ions scanned for each compound
was reduced to two in order to increase dwell time and therefore sensitivity. SIM windows,
fragmentor voltage, ions, and dwell times are listed in table 3.2.2.
28


Figure 3.3.2. TIC showing selected ion monitoring windows and fragmentor voltage setting.
Table 3.3.2. Selected ion monitoring windows, fragmentor voltage and ion assignments.
Time (Min.) Fragmentor Voltage Compound(s) Ion assignments Mass Analyzer Dwell time (ms/ion) Gain
2.00 80 Metformin 113.0, 130.1 439 2
5 80 Amoxicillin Cotinine 114.0, 208.0, 349.0 80.1,98.1, 177.1 145 2
6.3 70 Salbutamol 166.1,222.1,240.1 292 2
7.8 88 Cimetidine Acetaminophen Ranitidine Paraxanthine 159.0. 253.1 110.0, 152.0 176.0. 315.1 124.0, 181.1 108 2
13 90 Lisinopril 84.1,246.1,406.1 292 3
16 110 Caffeine Caffeine-3-13C 138.1, 195.1 139.1, 198.1 218 2
17.7 100 Enalaprilat Trimethoprim 230.1,303.1,349.1 206.1, 291.1 174 2
20 70 Digoxigenin 355.1,373.2, 391.2 292 2
21.7 100 Paroxetine metab. Phenacetin 13C Sulfamethoxazole 192.1.332.1 139.1, 181.1 156.1.254.1 145 2
22.85 110 Diltiazem Furosemide 178.1,415.1 352.9, 81.1 218 2
24.35 70 Fluoxetine 148.1,310.1 439 2
25.4 120 Dehydronifedipine 268.0, 284.0, 345.0 292 2
26.65 70 Warfarin 163.1,251.1,309.1 292 2
28.5 60 Ibuprofen 161.1,207.2 439 2
30 50 Gemfibrozil 205.2, 233.2, 273.0 292 2
29


3.4 Data Processing and Calibration Curves.
Data for quantitative analysis were acquired using the Hewlett-Packard Model 1100
HPLC-MS system and HP Chemstation software. Acquired data were then processed using
Target software. Five to seven point calibrations were used at the 0.010, 0.050, 0.100,
0.200, 0,400, 0.800, and 2.00 ng/pl levels on-column. The quantitative software computed
calibration curves for each compound. Curves were either linear or quadratic based on best
fit above a coefficient of regression of 0.995 or better. New calibration curves were obtained
for each sample run. Table 3.4.1 lists the number of points in the calibration curve, the type of
curve and the coefficient of regression for that compound for one of the sample runs.
Table 3.4.1. Calibration curve type and coefficient of regression for one calibration.
Compound Curve No. Points Cal Range ng/ul
Metformin Quad 7 0.010-2.0 0.99926
Cotinine Quad 7 0.010-2.0 0.99766
Amoxicillin Linear 6 0.050-2.0 0.99889
Salbutamol Quad 7 0.010-2.0 0.99770
Cimetidine Quad 7 0.010-2.0 0.99890
Acetaminophen Quad 7 0.010-2.0 0.99850
Ranitidine Linear 7 0.010-2.0 0.99958
Paraxanthine Linear 7 0.010-2.0 0.99837
Lisinopril Quad 5 0.10-2.0 0.99948
Caffeine Linear 7 0.010-2.0 0.99867
Enalaprilat Linear 6 0.050-2.0 0.99880
Trimethoprim Quad 7 0.010-2.0 0.99870
Digoxigenin Quad 7 0.010-2.0 0.99801
Paroxetine metabolite Quad 7 0.010-2.0 0.99954
Sulfamethoxazole Quad 7 0.010-2.0 0.99814
PhenacetinMC Quad 7 0.010-2.0 0.99884
Diltiazem Quad 7 0.010-2.0 0.99950
Furosemide Quad 5 0.10-2.0 0.99985
Fluoxetine Quad 7 0.010-2.0 0.99901
Dehydronifedipine Quad 7 0.010-2.0 0.99936
Warfarin Quad 7 0.010-2.0 0.99925
Ibuprofen Linear 7 0.010-2.0 0.99944
Gemfibrozil Linear 7 0.010-2.0 0.99917
30


3.5
Solid-phase Extraction.
3.5.1 Solid-phase sorbent trials.
Solid-phase extraction trials were performed on the four different sorbent types using
four varied solvent elution schemes. The results of the trials are listed in tables 3.5.1.1-
3.5.1.24 as the percent recovery, average percent recovery, standard deviation, and relative
standard deviation (RSD) of the determined recoveries for each trial.
Table 3.5.1.1. Percent recovery, carbopak eluted with methanol.
Carbopak MeOH
Compound Sample Sample Sample Sample Average Std Dev RSD
Acetaminophen 29 38 40 37 36 4.8 13
Amoxicillin 0 0 0 0 0 0.0 0
Cimetidine 0 3 4 4 2.8 1.9 69
Cotinine 119 206 111 214 163 55 34
Digoxigenin 0 3 0 0 0.75 1.5 200
Diltiazem 0 0 0 0 0 0.0 0
Enalaprilat 0 0.5 0 0.5 0.25 0.3 115
Fluoxetine 22 33 30 28 28 4.6 16
Furosemide 5 5 0 0 2.5 2.9 115
Gemfibrozil 0 0 0 0 0 0.0 0
O-Hydroxyhippuric acid 0 0 0 0 0 0.0 0
Ibuprofen 0 0 0 0 0 0.0 0
Lisinopril 0 0 0 0 0 0.0 0
Metformin 0 0 0 0 0 0.0 0
Paraxanthine 0 0 0 0 0 0.0 0
Paroxetine metabolite 0 0 0 0 0 0.0 0
Phenacetin 13C 0 0 0 0 0 0.0 0
Ranitidine 0 2 0 0 0.5 1.0 200
Salbutamol 10 8 4 8 7.5 2.5 34
Trimethoprim 21 45 28 36 33 10 32
Warfarin 0 0 0 0 0 0.0 0
(Phenacetin not in sample)
31


Table 3.5.1.2. Percent recovery, carbopak eluted with methanol pH 3.7.
Carbopak MeOH pH 3.7
Compound Sample Sample Sample Average Std Dev RSD
Acetaminophen 52 48 37 46 7.9 17
Amoxicillin 22 11 16 16 5.6 35
Cimetidine 55 55 58 56 1.7 3
Cotinine 89 113 97 100 12 12
Digoxigenin 8.0 4.0 0 4.0 4.0 100
Diltiazem 91 89 95 92 3.0 3
Enalaprilat 98 108 94 100 6.9 7
Fluoxetine 56 65 62 61 4.5 7
Furosemide 0 0 0 0 0.0 0
Gemfibrozil 2.0 2.0 2.0 2.0 0.0 0
O-Hydroxyhippuric acid 10 6.0 0 5.3 5.1 94
Ibuprofen 66 82 6.5 51 40 77
Lisinopril 75 69 79 74 5.1 7
Metformin 0 0 0 0 0.0 0
Paraxanthine 1.0 1.0 1.0 1.0 0.0 0
Paroxetine metabolite 0 0 0 0 0.0 0
Phenacetin 13C 0 0 0 0 0.0 0
Ranitidine 39 33 32 35 3.9 11
Salbutamol 6.0 14 9.0 10 4.0 42
Trimethoprim 105 117 107 110 6.3 6
Warfarin 13 10 7.0 10 3.0 30
(Phenacetin not in sample)
32


Table 3.5.1.3. Percent recovery, carbopak eluted with methanol/methanol pH 3.7.
Carbopak MeOH:MeOH pH 3.7
Compound Sample Sample Sample Average Std Dev RSD
Acetaminophen 32 19 8.0 20 12 62
Amoxicillin 0 0 0 0 0 0
Cimetidine 0 0 0 0 0 0
Cotinine 194 118 117 143 44 31
Digoxigenin 0 0 0 0 0 0
Diltiazem 0 0 0 0 0 0
Enalaprilat 0 0 0 0 0 0
Fluoxetine 25 12 20 19 6.7 35
Furosemide 0 0 0 0 0 0
Gemfibrozil 2 2 0 1.3 1.2 87
O-Hydroxyhippuric acid 0 0 0 0 0 0
Ibuprofen 0 0 0 0 0 0
Lisinopril 0 0 0 0 0 0
Metformin 0 0 0 0 0 0
Paraxanthine 0 0 0 0 0 0
Paroxetine metabolite 0 0 0 0 0 0
Phenacetin 13C 0 0 0 0 0 0
Ranitidine 0 0 0 0 0 0
Salbutamol 10 8.0 2.0 6.7 4.2 63
Trimethoprim 46 26 24 32 12 39
Warfarin 0 0 0 0 0 0
(Phenacetin not in sample)


Table 3.5.1.4. Percent recovery, carbopak eluted with methanol: acetonitrile (70:30).
Carbopak MeOH/ACN (70/30)
Compound Sample Sample Sample Sample Average Std Dev RSD
Acetaminophen 88 44 56 54 61 19 32
Amoxicillin 0 0 0 0 0 0 0
Cimetidine 5.0 1.0 7.0 2.0 4 2.8 73
Cotinine 119 70 87 81 89 21 24
Digoxigenin 43 38 13 10 26 17 65
Diltiazem 4.0 10 1.0 3.0 4.5 3.9 86
Enalaprilat 0 0 0 0 0 0 0
Fluoxetine 12 11 2.0 0 6.3 6.1 98
Furosemide 0 0 0 5.5 1.4 2.8 200
Gemfibrozil 0 0 0 0 0 0 0
O-Hydroxyhippuric acid 0 0 0 0 0 0 0
Ibuprofen 0 0 0 0 0 0 0
Lisinopril 0 0 0 0 0 0 0
Metformin 0 0 0 0 0 0 0
Paraxanthine 0 13 0 4.0 4.3 6.1 144
Paroxetine metabolite 0 0 0 0 0 0 0
Phenacetin 13C 0 0 0 0 0 0 0
Ranitidine 31 17 14 15 19 7.9 41
Salbutamol 8.0 3.0 14 3.0 7.0 5.2 75
Trimethoprim 66 38 66 41 53 15 29
Warfarin 0 0 0 0 0 0 0
(Phenacetin not in sample)
34


Table 3.5.1.5. Percent recovery, carbopak eluted with methanol: acetonitrile pH 3.7.
Carbopak MeOH/ACN pH 3.7
Compound Sample Sample Sample Average Std Dev RSD
Acetaminophen 63 101 189 118 65 55
Amoxicillin 28 20 25 24 4.0 17
Cimetidine 71 83 102 85 16 18
Cotinine 92 90 187 123 55 45
Digoxigenin 2.0 39 52 31 26 84
Diltiazem 144 143 284 190 81 43
Enalaprilat 109 114 144 122 19 15
Fluoxetine 62 69 103 78 22 28
Furosemide 0 0 5.0 1.7 2.9 173
Gemfibrozil 0 3.0 5.0 2.7 2.5 94
O-Hydroxyhippuric acid 43 63 95 67 26 39
Ibuprofen 91 108 167 122 40 33
Lisinopril 66 76 101 81 18 22
Metformin 0 0 1.0 0 0.6 173
Paraxanthine 0 8.0 8.0 5.3 4.6 87
Paroxetine metabolite 0 0 0 0 0 0
Phenacetin 13C 0 0 0 0 0 0
Ranitidine 55 65 73 64 9.0 14
Salbutamol 15 1.0 7.0 7.7 7.0 92
Trimethoprim 147 145 0 97 84 87
Warfarin 47 86 141 91 47 52
(Phenacetin not in sample)
35


Table 3.5.1.6. Percent recovery carbopak, eluted with methanol: acetonitrile/ methanol:
acetonitrile pH 3.7
Carbopak MeOH/ACN:MeOH/ACN pH 3.7
Compound Sample Sample Sample Average Std Dev RSD
Acetaminophen 111 36 49 65 40 61
Amoxicillin 17 11 9 12 4.2 34
Cimetidine 43 72 48 54 16 29
Cotinine 67 122 94 94 28 29
Digoxigenin 5.0 4.0 1.0 3.3 2.1 62
Diltiazem 112 218 145 158 54 34
Enalaprilat 20 39 23 27 10 38
Fluoxetine 36 54 43 44 9.1 20
Furosemide 0 5.5 0 1.8 3.2 173
Gemfibrozil 2.0 2.0 2.0 2.0 0 0
O-Hydroxyhippuric acid 0 1.0 0 0 0.6 173
Ibuprofen 60.5 145 90.5 99 43 43
Lisinopril 9 15.5 15 13 4 27
Metformin 0 0 0 0 0 0
Paraxanthine 0 2.0 1.0 1.0 1.0 100
Paroxetine metabolite 3.0 0 0 1.0 1.7 173
Phenacetin 13C 0 0 0 0 0 0
Ranitidine 10 39 24 24 15 60
Salbutamol 1.0 7.0 12 6.7 5.5 83
Trimethoprim 104 211 131 149 56 37
Warfarin 0 2.0 0 0.7 1.2 173
(Phenacetin not in sample)
36


Table 3.5.1.7. Percent recovery, HLB eluted with methanol.
HLB MeOH
Compound Sample Sample Sample Sample Average Std Dev RSD
Acetaminophen 92 80 83 98 88 8.3 9
Amoxicillin 0 0 0 0 0 0.0 0
Cimetidine 24 34 32 31 30 4.3 14
Cotinine 97 92 103 116 102 10 10
Digoxigenin 130 113 125 131 125 8.3 7
Diltiazem 94 78 77 82 83 7.8 9
Enalaprilat 8.5 7.5 8.0 8.5 8.1 0.48 6
Fluoxetine 50 41 34 59 46 11 24
Furosemide 87.5 77 83 86 83 4.6 5
Gemfibrozil 85 68 74 77 76 7.1 9
O-Hydroxyhippuric acid 19 19 22 18 20 1.7 9
Ibuprofen 119 97 114 116 111 9.8 9
Lisinopril 27 24 28 23 25 2.2 9
Metformin 1.0 1.0 0 1.0 0.8 0.5 67
Paraxanthine 103 88 95 100 97 6.6 7
Paroxetine metabolite 16 41 5.0 29 23 15.6 69
Phenacetin 13C 0 0 0 0 0 0.0 0
Ranitidine 54 49 51 56 53 3.1 6
Salbutamo! 108 92 102 117 105 11 10
Trimethoprim 111 90 98 104 101 8.9 9
Warfarin 127 106 118 116 117 8.6 7
(Phenacetin not in sample)
37


Table 3.5.1.8. Percent recovery, HLB eluted with methanol pH 3.7.
h LB MeOH pH 3.7
Compound Sample Sample Sample Average Std Dev RSD
Acetaminophen 98 88 93 93 5.0 5
Amoxicillin 12 4.0 4.5 6.8 4.5 66
Cimetidine 42 24 58 41 17 41
Cotinine 69 67 74 70 3.6 5
Digoxigenin 205 193 190 196 7.9 4
Diltiazem 93 92 103 96 6.1 6
Enalaprilat 13 12 13 12 0.3 2
Fluoxetine 71 70 82 74 6.7 9
Furosemide 67 55 60 60 6.0 10
Gemfibrozil 77 65 82 75 8.7 12
O-Hydroxyhippuric acid 25 35 33 31 5.3 17
Ibuprofen 66 79 81 75 8.1 11
Lisinopril 38 35 37 37 1.5 4
Metformin 0 0 0 0 0.0 0
Paraxanthine 105 93 98 99 6.0 6
Paroxetine metabolite 0 0 0 0 0.0 0
Phenacetin 13C 0 0 0 0 0.0 0
Ranitidine 53 46 65 55 9.6 18
Salbutamol 98 71 90 86 14 16
Trimethoprim 109 97 113 106 8.3 8
Warfarin 108 125 132 122 12 10
(Phenacetin not in sample)
38


Table 3.5.1.9. Percent recovery, HLB eluted with methanol/methanol pH 3.7.
HLB MeOH:MeOH pH 3.7
Compound Sample Sample Sample Average Std Dev RSD
Acetaminophen 72 74 88 78 8.7 11
Amoxicillin 0.50 0 0 0 0.29 173
Cimetidine 51 52 53 52 1.0 2
Cotinine 98 108 117 108 9.5 9
Digoxigenin 109 121 136 122 14 11
Diltiazem 98 95 105 99 5.1 5
Enalaprilat 15 9.0 11 12 2.8 24
Fluoxetine 82 74 78 78 4.0 5
Furosemide 53 66 76 65 12 18
Gemfibrozil 47 61 88 65 21 32
O-Hydroxyhippuric acid 10 8.0 11 10 1.5 16
Ibuprofen 54 58 83 65 16 24
Lisinopril 28 31 30 29 1.8 6
Metformin 1 3.0 3.0 2.3 1.2 49
Paraxanthine 102 105 108 105 3.0 3
Paroxetine metabolite 15 0 8.0 7.7 7.5 98
Phenacetin 13C 0 0 0 0 0.0 0
Ranitidine 43 48 70 54 14 27
Salbutamol 112 117 125 118 6.6 6
Trimethoprim 112 119 141 124 15 12
Warfarin 118 121 142 127 13 10
(Phenacetin not in sample)
39


Table 3.5.1.10. Percent recovery, HLB eluted with methanol: acetonitrile (70:30).
HLB MeOH/ACN (70/30)
Compound Sample Sample Sample Sample Average Std Dev RSD
Acetaminophen 90 105 85 79 90 11 13
Amoxicillin 0 1.4 2.0 0 0.84 1.0 120
Cimetidine 12 37 32 35 29 12 40
Cotinine 85 98 84 82 87 7.5 9
Digoxigenin 1.19 123 110 108 115 7.2 6
Diltiazem 94 86 79 75 83 8.5 10
Enalaprilat 10 6.8 7.5 0 6.1 4.3 70
Fluoxetine 59 46 41 45 48 7.8 16
Furosemide 87 103 89 0 70 47 67
Gemfibrozil 66 81 78 68 73 7.3 10
0-Hydroxyhippuric acid 28 27 26 0 20 14 67
Ibuprofen 97 111 102 82 98 12 12
Lisinopril 19 21 18.5 0 15 10 67
Metformin 0 0 0 0 0 0 0
Paraxanthine 91 100 73 75 85 13 15
Paroxetine metabolite 6.0 66 21 37 32 25 79
Phenacetin 13C 0 0 0 0 0 0 0
Ranitidine 54 71 63 55 61 7.8 13
Salbutamol 92 104 78 87 90 11 12
Trimethoprim 104 102 90 87 96 8.7 9
Warfarin 115 119 111 103 112 6.9 6
(Phenacetin not in sample)
40


Table 3.5.1.11. Percent recovery, HLB eluted with methanol: acetonitrile pH 3.7.
HLB MeOH/ACN pH 3.7
Compound Sample Sample Sample Average Std Dev RSD
Acetaminophen 99 87 76 87 12 13
Amoxicillin 0 0 0 0 0 0
Cimetidine 70 53 42 55 14 26
Cotinine 74 72 76 74 2.0 3
Digoxigenin 21 197 164 127 94 73
Diltiazem 11 94 100 68 50 73
Enalaprilat 0 0 0 0 0 0
Fluoxetine 97 82 92 90 7.6 8
Furosemide 0 0 0 0 0 0
Gemfibrozil 67 69 61 66 4.2 6
O-Hydroxyhippuric acid 0 0 0 0 0 0
Ibuprofen 80 70 64 71 8.4 12
Lisinopril 0 0 0 0 0 0
Metformin 0 0 0 0 0 0
Paraxanthine 99 93 95 96 3.1 3
Paroxetine metabolite 0 0 0 0 0 0
Phenacetin 13C 0 0 0 0 0 0
Ranitidine 66 48 36 50 15 30
Salbutamol 96 90 92 93 3.1 3
Trimethoprim 119 101 110 110 9.0 8
Warfarin 147 130 124 134 12 9
(Phenacetin not in sample)
41


Table 3.5.1.12. Percent recovery HLB, eluted with methanol: acetonitrile / methanol:
acetonitrile pH 3.7.
HLB MeOH/ACN:MeOH/ACN pH 3.7
Compound Sample Sample Sample Average Std Dev RSD
Acetaminophen 71 69 81 74 6.4 9
Amoxicillin 8.2 4.5 3.5 5.4 2.5 46
Cimetidine 42 36 52 43 8.1 19
Cotinine 78 78 102 86 14 16
Digoxigenin 114 110 144 123 19 15
Diltiazem 88 79 93 87 7.1 8
Enalaprilat 8.2 7.0 7.0 7.4 0.7 10
Fluoxetine 72 63 68 68 4.7 7
Furosemide 80 76 87 81 5.6 7
Gemfibrozil 68 48 68 61 12 19
O-Hydroxyhippuric acid 12 10 10 11 1.2 11
Ibuprofen 57 50 58 55 4.5 8
Lisinopril 31 28 29 29 1.5 5
Metformin 2.2 2.0 5.0 3.1 1.7 55
Paraxanthine 101 93 112 102 10 9
Paroxetine metabolite 2.2 7.0 0 3.1 3.6 117
Phenacetin 13C 0 0 0 0 0 0
Ranitidine 35 30 42 36 6.0 17
Salbutamol 96 93 117 102 13 13
Trimethoprim 111 99 120 110 11 10
Warfarin 119 106 135 120 15 12
(Phenacetin not in sample)
42


Table 3.5.1.13. Percent recovery, ENV+ eluted with methanol.
ENV+ MeOH
Compound Sample Sample Sample Sample Average Std Dev RSD
Acetaminophen 122 121 107 148 125 17 14
Amoxicillin 0 0 0 0 0 0 0
Cimetidine 0 0 0 0 0 0 0
Cotinine 113 104 91 125 108 14 13
Digoxigenin 134 115 98 129 119 16 14
Diltiazem 0 0 0 0 0 0 0
Enalaprilat 12 12 9 10.5 11 1.4 13
Fluoxetine 0 0 0 0 0 0 0
Furosemide 16 13 20.5 0 12 8.8 71
Gemfibrozil 30 25 15 17 22 7.0 32
O-Hydroxyhippuric acid 0 19 0 17 9 10 116
Ibuprofen 60 48 39 39 46 10 22
Lisinopril 0 0 0 0 0 0 0
Metformin 0 1 0 0 0 1 200
Paraxanthine 116 114 99 126 114 11 10
Paroxetine metabolite 0 0 0 0 0 0 0
Phenacetin 13C 0 0 0 0 0 0 0
Ranitidine 0 0 0 0 0 0 0
Salbutamol 0 0 0 0 0 0 0
Trimethoprim 0 0 0 0 0 0 0
Warfarin 8.0 7.0 5.0 4.0 6.0 1.8 30
(Phenacetin not in sample)
43


Table 3.5.1.14. Percent recovery, ENV+ eluted with methanol pH 3.7.
ENV+ MeOH pH 3.7
Compound Sample Sample Sample Average Std Dev RSD
Acetaminophen 134 120 227 160 58 36
Amoxicillin 14 10 0 8 7.2 90
Cimetidine 13 15 16 15 1.5 10
Cotinine 109 97 203 136 58 43
Digoxigenin 132 120 274 175 86 49
Diltiazem 65 57 108 77 27 36
Enalaprilat 17 18.5 32 22 8.0 36
Fluoxetine 55 58 118 77 36 46
Furosemide 50 46 66 54 11 20
Gemfibrozil 37 22 52 37 15 41
O-Hydroxyhippuric acid 32 33 56 40 14 34
Ibuprofen 74.5 59 109 81 25 31
Lisinopril 44 37 55 45 8.8 20
Metformin 102 92 176 123 46 37
Paraxanthine 123 116 185 141 38 27
Paroxetine metabolite 48 43 45 45 2.5 6
Phenacetin 13C 0 0 0 0 0.0 0
Ranitidine 10 7 12 10 2.5 26
Salbutamol 123 112 223 153 61 40
Trimethoprim 107 98 208 138 61 44
Warfarin 15 11 23 16 6.1 37
(Phenacetin not in sample)
44


Table 3.5.1.15. Percent recovery, ENV+ eluted with methanol/methanol pH 3.7.
ENV+ MeOH:MeOH pH 3.7
Compound Sample Sample Sample Average Std Dev RSD
Acetaminophen 204 94 164 154 56 36
Amoxicillin 0 6.5 4.5 3.7 3.3 91
Cimetidine 0 0 0 0 0 0
Cotinine 146 79 134 120 36 30
Digoxigenin 160 80 133 124 41 33
Diltiazem 0 0 48 16 28 173
Enalaprilat 31 0 0 10 18 173
Fluoxetine 0 26 34 20 18 89
Furosemide 0 3 5.5 2.8 2.8 97
Gemfibrozil 16 28 49 31 17 54
O-Hydroxyhippuric acid 59 34 62 52 15 30
Ibuprofen 41 0 0 14 23 173
Lisinopril 0 0 0 0 0 0
Metformin 0 88 0 29 51 173
Paraxanthine 161 0 140 100 88 87
Paroxetine metabolite 0 0 0 0 0 0
Phenacetin 13C 0 0 0 0 0 0
Ranitidine 0 0 0 0 0 0
Salbutamol 7.0 0 0 2.3 4.0 173
Trimethoprim 0 0 0 0 0 0
Warfarin 5.0 0 2.0 2.3 2.5 108
(Phenacetin not in sample)


Table 3.5.1.16. Percent recovery, ENV+ eluted with methanol: acetonitrile (70:30).
ENV+ MeOH/ACN (70/30)
Compound Sample Sample Sample Sample Average Std Dev RSD
Acetaminophen 81 118 94 111 101 17 17
Amoxicillin 13 5.5 6.5 6.5 7.8 3.2 41
Cimetidine 0 0 3.0 0 0.8 1.5 200
Cotinine 50 87 71 86 74 17 24
Digoxigenin 76 114 101 110 100 17 17
Diltiazem 0 0 4.0 0 1.0 2.0 200
Enalaprilat 8.0 9.0 8.5 9.0 8.6 0 6
Fluoxetine 0 0 9.0 0 2.3 4.5 200
Furosemide 36 35 77 33 45 21 47
Gemfibrozil 41 32 47 50 43 7.9 19
O-Hydroxyhippuric acid 26 29 37 30 31 4.7 15
Ibuprofen 53 56 66 62 59 5.9 10
Lisinopril 10 4.5 15 5 8.6 4.9 57
Metformin 12 0 80 0 23 38 167
Paraxanthine 75 106 97 116 99 17 18
Paroxetine metabolite 0 0 0 0 0 0 0
Phenacetin 13C 0 0 0 0 0 0 0
Ranitidine 0 0 0 0 0 0 0
Salbutamol 0 0 57 0 14 29 200
Trimethoprim 0 0 0 0 0 0 0
Warfarin 56 48 89 63 64 18 28
(Phenacetin not in sample)
46


Table 3.5.1.17. Percent recovery, ENV+ eluted with methanol: acetonitrile pH 3.7.
ENV+ MeOH/ACN pH 3.7
Compound Sample Sample Sample Average Std Dev RSD
Acetaminophen 81 67 74 10 13
Amoxicillin 12 24 8.5 15 7.9 55
Cimetidine 14 51 19 28 20 72
Cotinine 48 135 39 74 53 72
Digoxigenin 105 296 86 162 116 72
Diltiazem 75 277 77 143 116 81
Enalaprilat 23 52.5 28 34 16 46
Fluoxetine 81 78 80 2.1 3
Furosemide 71 116 69 85 27 32
Gemfibrozil 50 118 40 69 42 61
O-Hydroxyhippuric acid 45 98 45 63 31 49
I bu profen 47 131 40 73 51 70
Lisinopril 60 130 72 87 37 43
Metformin 64 164 55 94 61 64
Paraxanthine 88 231 77 132 86 65
Paroxetine metabolite 0 0 0 0 0 0
Phenacetin 13C 0 0 0 0 0 0
Ranitidine 16 45 19 27 16 60
Salbutamol 69 219 57 115 90 78
Trimethoprim 84 82 83 1 2
Warfarin 88 277 81 149 111 75
(Phenacetin not in sample)
47


Table 3.5.1.18. Percent recovery ENV+, eluted with methanol: acetonitrile/ methanol:
acetonitrile pH 3.7.
ENV+ MeOH/ACN:MeOH/ACN pH 3.7
Compound Sample Sample Sample Average Std Dev RSD
Acetaminophen 102 88 95 10 10
Amoxicillin 12 16 29 19 8.6 46
Cimetidine 20 17 38 25 11 45
Cotinine 66 58 143 89 47 53
Digoxigenin 97 81 236 138 85 62
Diltiazem 72 57 142 90 45 50
Enalaprilat 19 19 39 25 12 47
Fluoxetine 63 53 137 84 46 54
Furosemide 72 74 109 85 21 25
Gemfibrozil 26 23 38 29 7.9 27
O-Hydroxyhippuric acid 52 47 94 64 26 40
Ibuprofen 37 40 79 52 23 45
Lisinopril 39 33 73 48 21 44
Metformin 81 70 171 107 55 52
Paraxanthine 99 93 206 133 64 48
Paroxetine metabolite 14 12 29 18 9.3 51
Phenacetin 13C 0 0 0 0 0 0
Ranitidine 15 14 41 23 15 66
Salbutamol 90 75 240 135 91 68
Trimethoprim 92 71 224 129 83 64
Warfarin 69 56 131 85 40 47
(Phenacetin not in sample)
48


Table 3.5.1.19. Percent recovery, C-18 eluted with methanol.
C-18 MeOH
Compound Sample Sample Sample Sample Average Std Dev RSD
Acetaminophen 0 0 0 2.0 0.5 1.0 200
Amoxicillin 0 0 0 0 0 0.0 0
Cimetidine 44 143 107 98 50 51
Cotinine 79 184 136 133 53 40
Digoxigenin 80 155 123 270 157 81 52
Diltiazem 0 7.0 12 25 11 11 96
Enalaprilat 0 0 0 0 0 0.0 0
Fluoxetine 0 0 12 17 7.3 8.6 119
Furosemide 5.0 6.0 5.5 6 5.6 0.5 9
Gemfibrozil 48 100 88 169 101 50 50
O-Hydroxyhippuric acid 0 0 0 0 0 0.0 0
I bu profen 33 60 70 152 79 51.0 65
Lisinopril 4.5 8.5 9.5 15 9.4 4.3 46
Metformin 13 36 48 95 48 35 72
Paraxanthine 11 18 16 31 19 8.5 45
Paroxetine metabolite 7.0 20 49 10 22 19 89
Phenacetin 13C 59 133 112 101 38 38
Ranitidine 0 2.0 4.0 6.0 3.0 2.6 86
Salbutamol 72 174 136 127 52 40
Trimethoprim 52 122 114 96 38 40
Warfarin 96 217 162 158 61 38
49


Table 3.5.1.20. Percent recovery, C-18 eluted with methanol pH 3.7.
C-18 MeOH pH 3.7
Compound Sample Sample Sample Average Std Dev RSD
Acetaminophen 0 0 0 0 0 0
Amoxicillin 16 14 6 12 5.3 44
Cimetidine 139 107 72 106 34 32
Cotinine 95 80 64 80 16 19
Digoxigenin 155 124 91 123 32 26
Diltiazem 147 103 91 114 29 26
Enalapriiat 0 0 0 0 0 0
Fluoxetine 105 71 61 79 23 29
Furosemide 7.0 5.5 6 6 0.8 12
Gemfibrozil 124 109 78 104 23 23
O-Hydroxyhippuric acid 0 0 0 0 0 0
Ibuprofen 112 94 69 91 22 24
Lisinopril 14 17 12 14 2.8 20
Metformin 83 66 53 67 15 22
Paraxanthine 26 22 14 21 6.1 30
Paroxetine metabolite 35 43 94 57 32 56
Phenacetin 13C 191 141 101 144 45 31
Ranitidine 103 74 49 75 27 36
Salbutamol 15 119 87 74 53 72
Trimethoprim 153 108 95 119 30 26
Warfarin 218 152 112 161 54 33


Table 3.5.1.21. Percent recovery, C-18 eluted with methanol/methanol pH 3.7.
C-18 MeOH:MeOH pH 3.7
Compound Sample Sample Sample Average Std Dev RSD
Acetaminophen 0 0 1.0 0 0.6 173
Amoxicillin 1.5 7.5 7.0 5.3 3.3 62
Cimetidine 84 104 153 114 36 31
Cotinine 77 91 128 99 26 27
Digoxigenin 97 126 185 136 45 33
Diltiazem 85 107 142 111 29 26
Enalaprilat 0 0 0 0 0 0
Fluoxetine 69 81 88 79 10 12
Furosemide 5.5 5.5 6.0 5.7 0.29 5
Gemfibrozil 75 83 109 89 18 20
O-Hydroxyhippuric acid 0 0 0 0 0 0
Ibuprofen 62 78 112 84 26 30
Lisinopril 10 10 13 11 1.7 16
Metformin 52 68 83 68 16 23
Paraxanthine 13 18 20 17 3.6 21
Paroxetine metabolite 40 25 11 25 15 57
Phenacetin 13C 105 146 272 174 87 50
Ranitidine 44 51 62 52 9.1 17
Salbutamol 91 119 172 127 41 32
Trimethoprim 96 125 178 133 42 31
Warfarin 116 146 229 164 59 36
51


Table 3.5.1.22. Percent recovery, C-18 eluted with methanol: acetonitrile (70:30).
C-18 MeOH/ACN (70/30)
Compound Sample Sample Sample Sample Average Std Dev RSD
Acetaminophen 1.0 0 1.0 0 0.5 0.6 115
Amoxicillin 0 3.5 1.0 0 1.1 1.7 147
Cimetidine 46 88 69 19 56 30 54
Cotinine 136 98 117 27 23
Digoxigenin 145 98 122 33 27
Diltiazem 0 25 0 0 6.3 13 200
Enalaprilat 0 0 0 0 0 0 0
Fluoxetine 0 3 0 0 0.8 1.5 200
Furosemide 0 5.5 0 0 1.4 2.8 200
Gemfibrozil 161 109 140 63 118 43 36
O-Hydroxyhippuric acid 0 0 0 0 0 0 0
Ibuprofen 173 93 173 73 128 53 41
Lisinopril 0 0 0 0 0 0 0
Metformin 27 47 30 14 30 14 46
Paraxanthine 24 18 27 14 21 6 28
Paroxetine metabolite 0 1 0 0 0 0.5 200
Phenacetin 13C 139 92 116 33 29
Ranitidine 0 11 0 0 2.8 5.5 200
Salbutamol 14 80 13 5.0 28 35 125
Trimethoprim 1.0 64 0 0 16 32 196
Warfarin 272 164 0 109 136 113 83
52


Table 3.5.1.23. Percent recovery C-18, eluted with methanol:acetonitrile pH 3.7.
C-18 MeOH/ACN pH 3.7
Compound Sample Sample Sample Average Std Dev RSD
Acetaminophen 1.0 3.0 6.0 3.3 2.5 75
Amoxicillin 18 25 26 23 4.2 18
Cimetidine 113 122 167 134 29 22
Cotinine 97 100 163 120 37 31
Digoxigenin 137 142 208 162 40 24
Diltiazem 131 133 280 181 85 47
Enalaprilat 0 0 0 0 0 0
Fluoxetine 97 105 187 130 50 38
Furosemide 6 3 5.5 4.8 1.6 33
Gemfibrozil 95 100 94 96 3.2 3
O-Hydroxyhippuric acid 0 0 0 0 0 0
Ibuprofen 126 142 242 170 63 37
Lisinopril 13 13 18 14 3.2 22
Metformin 79 92 132 101 28 27
Paraxanthine 20 23 31 25 5.7 23
Paroxetine metabolite 35 94 27 52 37 70
Phenacetin 13C 146 180 163 24 15
Ranitidine 92 93 79 88 7.8 9
Salbutamol 138 143 141 3.5 3
Trimethoprim 134 140 137 4.2 3
Warfarin 174 200 273 216 51 24
53


Table 3.5.1.24. Percent recovery C-18, eluted with methanol: acetonitrile/methanol:
acetonitrile pH 3.7.
C-18 MeOH/ACN:MeOH/ACN pH 3.7
Compound Sample Sample Sample Average Std Dev RSD
Acetaminophen 1.0 2.0 0 1.0 1.0 100
Amoxicillin 6.0 8.5 4.0 6.2 2.2 36
Cimetidine 94 136 70 100 33 33
Cotinine 96 185 76 119 58 49
Digoxigenin 117 191 95 134 50 37
Diltiazem 113 202 89 135 59 44
Enalapriiat 0 0 0 0 0 0
Fluoxetine 87 150 77 105 40 38
Furosemide 5.5 0 0 1.8 3.2 173
Gemfibrozil 90 122 69 94 27 28
O-Hydroxyhippuric acid 0 0 0 0 0 0
Ibuprofen 98 191 88 126 57 45
Lisinopril 8.5 17 10 12 4.2 36
Metformin 68 118 58 81 32 39
Paraxanthine 19 27 15 20 6.1 30
Paroxetine metabolite 11 0 0 3.7 6.4 173
Phenacetin 13C 131 103 117 20 17
Ranitidine 47 95 40 61 30 49
Salbutamol 120 178 80 126 49 39
Trimethoprim 112 91 102 15 14
Warfarin 148 276 113 179 86 48
The results of the SPE trials were compared for average recovery and standard deviation.
Samples with erroneously high recoveries and high standard deviation were subjected to Q
tests at the 95 % confidence level. A sample containing more than three failed Q tests was
removed and the average for that trial recomputed. The average and standard deviation
comparisons are displayed for each cartridge for the six different elution solvent systems in
figures 3.5.1.1 3.5.1.11.
54


£*
o
>
o
o
0>
o'
200
180
160
140
120
100
80
60
40
20
0
Average recovery, Carbopak




t JL ;

dm



^ - <& *?SnpS* O^* -<
§'


>V
!; MeOH
! MeOH acid
i MeOH: N/A
ACN/MeOH
AON acid
AON: N/A
Figure 3.5.1.1. Average recovery, Carbopak SPE cartridges. (ACN/MeOH is 30:70; N/A
refers to neutral followed by acidic solvent.)
55


Adjusted average recovery, Carbopak
180
160
140
120
I 100
80
MeOH
MeOH acid
MeOH: N/A
ACN/MeOH
AON acid
AON: N/A
Figure 3.5.1.2. Carbopak SPE cartridge average recovery adjusted for sample in ACN acid.
Standard Deviation, Carbopak Recovery

!
w
90
80
70
60
50
40
30
20
10
0



n


r II 1 J
rl lid I J i sf 1.1 tf nm j1_. JL 1 ft. n rfftlil


j/


r &
/>
MeOH
MeOH acid
MeOH: N/A
ACN/MeOH
ACN acid
ACN: N/A
Figure 3.5.1.3. Standard deviation, Carbopak SPE average recovery.
56


Average recovery, HLB
MeOH
MeOH acid
MeOH: N/A
ACN:MeOH
AON acid
AON: N/A
Figure 3.5.1.4. Average recovery HLB SPE cartridges.
Standard deviation, HLB recovery
. j/. : t A'
^ JU .2 an - . w. -vj. -.V n i o MeOH 1 MeOH acid MeOH: N/A AON: MeOH
.5 '4
s 70 fin - l3 ; v i ,* t if'* v V
o DU o 0\j a - i ; *. -J £[* .*3Jrfr -,'f. 4 *. : i A A<\
. "t V \ J * AON acid
£ CO w 10 - K jr iT1*!! 11 n [) (i nf rl* 0 -v.n ! ACN: N/A
111 1 U I 1 Ji. killfl ii fa:ik I r i IIIlH
<-N ^ iN A ^ Js Js .is cs ,$s .0 C> j&s / /'
Figure 3.5.1.5. Standard deviation, HLB SPE average recovery.
57


Figure 3.5.1.6. Average recovery, ENV+ SPE cartridges.
Figure 3.5.1.7. ENV+ SPE cartridge average recovery adjusted for sample in MeOH acid.
58


Standard deviation, ENV+ recovery
Figure 3.5.1.8. Standard deviation, ENV+ average recovery.
Figure 3.5.1.9. Average recovery, C-18 SPE cartridges.
59


Figure 3.5.1.10. C-18 SPE cartridge average recovery adjusted for sample in ACN acid.
C
o
re
>
! ' *o
re
TJ
c
re
**
o
a
E
re
co
120
100
80
60
40
20
0
Jifc.

Standard deviation, C-18 recovery
1
nfl

i
Ikfi. 1 M L

II,
/?//&////
w v v>vv^

4
&

MeOH
B MeOH acid
MeOH: N/A
ACN/MeOH
B ACN acid
O ACN: N/A

Figure 3.5.1.11. Standard deviation, C-18 average recovery.
60


The highest recovery by total percent for each type of SPE sorbent and elution
solvent system was then compared among sorbent types for percent recovery and standard
deviation of measurements (figures 3.5.1.12-3.5.1.15).
Figure 3.5.1.12. Highest recovery for Carbopak SPE cartridges, acidic MeOH: acetonitrile.
61


C-18 MeOH/ACN pH 3.7
Figure 3.5.1.13. Highest recovery for C-18 SPE cartridges, acidic methanol/acetonitrile.
ENV+ MeOH/ACN pH 3.7
180
160
140
Figure 3.5.1.14. Highest recovery for ENV+ SPE cartridges, acidic methanol/acetonitrile.
62


Figure 3.5.1.15. Highest recovery for HLB SPE cartridges, methanol/acidic methanol.
3.5.2 SPE Break-through.
Tests for break through for each type of cartridge yielded zero recovery for any
compounds. In addition, analysis of the 1 ml, 5% methanol cartridge wash also yielded zero
recovery of any compounds.
3.6 Method Detection Level
The MDL was determined by spiking pristine mountain water with concentrations
thought to be about five times higher than the expected MDL. Two trials were needed, as the
MDL was high for a small number of compounds. For most of the compounds water was
spiked at the 100 part per trillion level and for the other compounds at one part per billion.
The results of the trials are combined and listed in table 3.6.1.
63


Table 3.6.1. MDL in ng/ml on-column amount (equivalent to jj.g/1 for samples).
Compound MDL (ng/pl) on-column
Metformin 0.0034
Cotinine 0.023
Amoxicillin ND*
Salbutamol 0.030
Cimetidine 0.0067
Acetaminophen 0.0086
Ranitidine 0.010
Paraxanthine 0.019
Lisinopril ND
Caffeine 0.014
Enalaprilat 0.15
Trimethoprim 0.014
Digoxigenin 0.0077
Paroxetine metabolite ND
Sulfamethoxazole 0.023
Phenacetin 13C 0.013
Diltiazem 0.012
Furosemide ND
Fluoxetine 0.018
Dehydronifedipine 0.0095
Warfarin 0.0061
Ibuprofen 0.018
Gemfibrozil 0.015
*Not detected under experimental conditions.
3.7 Environmental Sample Analysis
Seventy-seven samples were collected by US Geological Survey personnel and
shipped overnight to the testing facility. Unfiltered samples were filtered upon receipt, and all
samples were refrigerated at 4C until extraction. Samples that showed signs of microbial
growth While in storage were re-filtered prior to extraction. Samples were extracted using
HLB extraction cartridges, eluted with neutral and acidified methanol, filtered, and vialed
according to methods described in section 2.3. Data was acquired as described in section
64


2.2. Each analytical run of samples included a fresh calibration set. An E marked
detections that were below the MDL but that clearly showed peaks that were out of the noise,
with the appropriate ions of correct relative abundance. Each analytical set included
continuing calibration verification samples at the 0.100 and 0.800 or 1.00 part per billion
levels. Set spikes were 1.00 part per billion concentration for a recovery of 100%. Results of
sample analysis are listed in tables 3.7.1 3.7.8. A summary of sample set results is listed in
table 3.7.9 and the spike recoveries for each sample set are summarized in table 3.7.10.
65


Table 3.7.1. Sample set number 10-5 results.
MRDP Schedule 9003
Sample Set Report
Set Number lib ID Project Account Station ID Station Name Lab ID Projoct Account Station ID Station Nam* Lab ID Projoct Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station (D Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Set Blank Lib ID Set Spike Lab ID CCV
[10-5] 992730154 451313500 341145064111201 City of Cummings WPCP.GA 451313600 02335540 Kelly Mill Branch Tnbutary at Castleberry Road, £A 992730156 451313600 02335741 Big Creek below Waterworks Intakes (City of Roswell), GA 992740075 451310200 335640064252601 Cobb Counly Waler Intake, GA- Laboratory Spike 992740076 451313600 335640064252601 Cobb Counly Water Intake. GA 992740077 451313600 334939084271501 Atlanta Waler Works Intake, GA 992740078 451313600 334918064270601 Atlanta Waler Works Chattahoochee Water Treatment Plant, GA 992740114 451313600 340106064210601 City of Roswell Water Treatment Plant. GA 992740115 451313600 340003064232401 Big Creek WPCP. GA 992740116 451313600 335640064241901 Cobb Counly Waler Treatmenl Planl.GA 992788040 992789040
Collection Date 09/79*9 09/29/99 09/29/99 09/30/99 09/3399 09/30/99 09/3099 09/3099 Q9/3099 09/3099
Collection Timo i 15 1230 1115 1110 1100 1245 1400 0900 1930 1030
Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample
Compound Volume Cone Volume Cone Volume Cone Volume Cone Volume Cone Volume Cone Volume Cone Volume Cone Volume Cone Cone Cone Cone *
(ml) W( (ml) Wt (ml) wo M Wt (ml) (ugty (ml) (W* (ml) (ml) (poty (ml) w (ml) 0*941 0*94 ng/pl ng/p!
67069 864 85 86309 87193 86015 882 97 675 42 916 48 300 05 900 82
Metformin 0 0 0 0 0 0 0 0 E 00732 0 0 0 0 0654 0669
Amoxicillin 0 0 0 0 0 0 0 0 0 0 0 0 0 066 0577
Cotinine 0 00483 0 0871 0 0 0 0 0 0 0 0 667 0 0616 0982
Salbutamol 0 0 0 0825 0 0 0 0 0 0 0 0584 0 0697 0 475
Cimetidine 0 0 0 0679 0 0 0 0 0604 0 0 0 312 00656 0 540
Acetaminophen 0 0 0436 0 0 802 0 E 000272 0 0 0 0 f 000962 0 553 0.110 0 600
Ranitidine 0 0 0 0391 0 0 0 0 0 0 0 0286 0 0643 0 354
Paraxanthine 0 0 0601 0 1.05 0 0 0 0 0 0 0 0 799 0105 0834
Lisinopril 0 0 0 0 0 0 0 0 0 0 0 0318 0122 111
Enalaprilat 0 0 0 0 545 0 0 0 0 0 0 0 0110 0111 105
Trimethoprim E 00140 0 0 0.796 0 0 0 0 0 0797 0 0 0 749 0 0913 0833
Diqoxiflenin 0 0 0 0 327 0 0 0 0 0 0 0 0 793 0106 0 796
Paroxetine Metabolite 0 0 0 0 0 0 0 0 0 0 0 0 00506 0811
Diltiazem 00163 0 0 0 526 E 0 00397 E 000444 0 0 00612 0 0 0 701 0 0916 0 857
Furosemide 0 0 0 0 0 0 0 0 0 0 0 0527 0 129
Fluoxetine 0 0 0 0 0 0 0 0 0 0 0 0759 0 0603 0 790
Warfarin 0 0 0 0052 0 0 0 0 0 0 0 0 851 0 0955 0832
Ibuprofen 0 0 0 0 754 0 0 0 0 0 0 0 0 719 0 0991 0 955
Gemfibrozil 0 0 0 0497 0 0 0 0 0 0 0 0644 00662 0 982
Caffeine 0 0 639 0114 1.05 00535 00564 0 0530000 0 150000 0 00633 0 0 628 0109 0987
Sulfamethoxazole 0 234 0 0 0665 E 0 00905 0 0 0 0 226 0 0 0815 0105 0604
Dehydronifedipine 0 0 0 1.23 0 0 0 0 0 0 0 0902 00925 0813
Caffeine13CISTD 1.15 1 16 1 16 1 15 1 16 1 13 1 14 1 09 1 14 111 1 15 l 1 1
Phenacetin13C 0 341 0297 0 384 0560 0559 0 535 0 562 0365 0 405 0566 1.13 0 953 0 0923 0 736
100% = spike recmery
E = estrmaled ( ** Lab Spike


Table 3.7.2. Sample set number 9279.10 results.
Set Number Lab ID Project Account Station ID Station Namo Lab ID Project Account Station ID Station Namo Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Namo Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Namo Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Set Blink Lab ID Set Spike Lab ID CCV
9279.10 991030160 466319401 06451210 S F Iowa Rfitf NE NewPromdence, IA 991040074 462911860 365618094013101 Clear Ck below Pierce City 991040075 488319401 365618094013101 Clear Ck below Pierce City 991060003 07189540 Cave Spgs BR- SW City Mo 991120001 488319401 07373420 Mississippi R. at Si Ftancisville 991130098 4/0652421 340127117355901 Upland Sewage Outflow toCutaianga 991130348 488319401 394938104565300 S Platte abv Clear Creek nrCommerce cm 991250010 488319401 06893560 Brush Creek At Kansas City 991270050 488319481 12128000 Thornton Ck 991310005 488319401 07241550 N Canadmo R nr Mar*ah Ok 992790040 992799040
Collection Date 04*8*9 044)7*9 044)7*9 04*7*9 04/19*9 04/15*9 04/21/99 04/29*9 05*6*9 05*3*9
Collection Time 1000 0640 0640 1000 1130 1700 1250 1245 1050 1105
Compound Sample Volume (ml) Sample Cone Sample Volume (ml) Sample Cone M Sample Volume H Sample Cone M Sample Volume M Sample Cone m Sample Volume M Sample Cone M Sample Volume (ml) Semple Cone Sample Volume (ml) Sample Cone (499 Sample Volume (ml) Sample Cone M Sample Volume (ml) Sample Cone <49* Sample Volume (ml) Semple Cone m Sample Cone ((.gflO Sample Cone* Wpl) 0.100 ng/pJ 1JM ng/pt
934 04 826 26 655 90 89011 83961 81992 8/1 14 820 46 66889 87089
Metformin 0 0 0 0 0 0 0 0 0 0 0 0 0 0975 0892
Amoxicillin 0 0 0 0 0 0 0 0 0 0 0 0 0107 0 744
Cotinine 0 E 00199 E 00169 0 0 0 0476 0 896 0 222 0 E 0013 0 0 761 0088 0 778
Salbutamol 0 0 0 0 0 0 0 0 0 0 0 0 754 0104 0935
Cimeddine 0 0 00119 0 0 0124 0 0 0 0 0 0 305 00922 0.B18
Acetaminophen 0016 0 0 0 0 0 0 E 100 0 0 0 0692 0118 0 968
Ranitidine 0 0 0 0 0 0 0 0 0 0 0 0 387 0 0073 ' 0937
Paraxanthine 0 0 0 0 0 0 00526 19 0 0 0 0995 0116 0 973
Lisinopril 0 0 0 0 0 0 0 0 0 0 0 0441 0 0941
Enalaprilat 0 0 0 0 0 0 0 0 0 0 0 0125 0129 104
Trimethoprim 0 E 0.0066 E 00056 0 0 00584 0 303 0 0232 0 0 0 0 846 0103 0 847
Oigoxipenin 0 0 0 0 0 0 0 0 0 0 0 0863 0105 0 849
Paroxetine Metabolite 0 0 0 0 0 0 0 0 0 0 0 0 0943 00003 0 664
Diltiazem 0 0 0 0 0 0 00489 0 0 0 0 0733 00926 0838
Furosemide 0 0 0 0 0 0 0 0 0 0 0 0648 0111 0932
Fluoxetine 0 0 0 0 0 0 0 0 0 0 0 0 932 0102 0917
Warfarin 0 0 0 0 0 0 0 0 0 0 0 0 890 00987 0 857
Ibuprofen 0 0 0 0 0 0 0 105 0 0 0 0 754 00942 0.876
Gemfibrozil 0 0 0 0 0 0 0 786 0 0 0 0 0 766 0 0828 0 959
Caffeine 00699 0 0791 0066 0 0 0681 0114 0 604 0169 00676 0 0965 0102 0876
Sulfamethoxazole 0 0 0 0 0 0 0 0 0 0 0 0957 0107 0.896
Dehydronifedipine 0 E 00048 E 000577 0 0 00295 0 0 0 0 0 0962 0 0948 0851
Caffeinel 3CISTD 107 121 1 17 112 1 19 1.22 1 15 122 1 15 VIS 121 1 1 1
Phenacetinl 3C 0 565 0773 0752 0 647 0 456 0 57 0384 104 0473 0394 155 1094 0104 079
* x 100% = spike recovery
E = estimated (

Table 3.7.3. Sample set number 9279.11 results.
Set Number Lib ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Set Blank Lab ID Set Spike Lab ID CCV
9279.11 992420034 451313600 34010808421000! Oily of Roswell Water Treatment Plant, GA 992420035 451313600 340003064232401 City of Cummings WPCP, GA 997420038 451313600 02335540 Kelly Mil! Blanch Tributary at Castleberry Road. 992700004 470600356 11274538 Orestimba Cr Nr Ciows Landing 992700005 470648500 11303500 SJR NrVemahs 992700023 470600356 11262900 Mud Slough Nr Gustine CA 992700024 470600356 11274560 nos 992700025 470600356 55565555 French Camp Slough 992730152 451313600 335732084153401 Crocked Creek WPCP, GA 992730153 451313600 33583708417310! Johns Creek WPCP, GA 992798041 992799041
Collection Date 06727799 09427/99 09/27499 mm 09/22499 09421/99 mm 09/22r99 09/2999 09/29.99
Collection Time 1145 1400 1430 1800 1120 1110 1440 1320 0845 0930
Compound Sample Volume M Sample Cone wo Semple Volume (ml) Sample Cone WJ Sample Volume M Sample Cone wo Sample Volume (ml) Sample Cone Wi Semple Volume H Sample Cone w Sample Volume (ml) Sample Cone wo Sample Volume (ml) Sample Cone Wti Sample Volume (ml) Sample Cone wo Sample Volume M Sample Cone wo Sample Volume (ml) Sample Cone wo Sample Cone wo Sample Cone* wo 0.100 ng//d 1JM
889.038 855 40 880.57 695.91 75292 79197 838 74 74348 869.50 853 74
Metformin 0 0 0 0 0 0 0 0 0 0 0 000906 00936 0822
Amoxicillin 0 0 0 0 0 0 0 0 0 0 0 0 000 0 0766 0505
Cotinine 0 0 E 00175 0 0 0 E 00113 0 0 0 0 0 723 00806 0 699
Salbutamol 0 0 0 0 0 0 0 0 0 0 0 0 659 00971 0861
Cimetidine 0 0 0 0 0 0 0 0 0.670 0 0 0.258 0.0346 0756
Acetaminophen 0 0 0423 E 0 00392 E 0.00400 0 E 000573 0 0 0 0 0.761 0132 0979
Ranitidine 0 0 0 0 0 0 0 0 0 0 0 0 353 00836 0 859
Paraxanthine 0 0 0193 0 0 0 0 0273 0 0 0 0 0 999 0137 104
Lisinopril 0 0 0 0 Q 0 0 0 O 0 0 0.446 0.124 1.09
Enalaprilat 0 0 0 0 0 0 0 0 0 0 0 0.172 0.157 115
Trimethoprim 0 0 E 00125 0 0 0 0 0241 0 0 0 0458 0 0 795 0 0919 0810
Diqoxigenin 0 0 0 0 0 0 0 0 0 0 0 0911 0115 0872
Paroxetine Metabolite 0 0 0 0 0 0 0 0 0 0 0 0.025 0.0316 0786
Diltiazem 0 0.0100 0 0 0 0 0 0 00277 E 000243 0 0677 0.0842 0793
Furosemide 0 0 0 0 0 0 0 0 0 0 0 0 629 0122 100
Fluoxetine 0 0 0 0 0 0 0 0 0 0 0 0842 0 0915 0855
Warfarin 0 0 0 0 0 0 0 0 0 0 0 0867 0.0975 0.808
Ibuprofen 0 0 0 0 0 0 00941 0 0 0 0 0789 0 0974 0912
Gemfibrozil 0 0 0 0 0 0 0139 0 0 0 0 082) 00958 0996
Caffeine 00557 0 0233 0 0 0 0175 0 0 0 0 0888 0106 0867
Sulfamethoxazole 0 0 0 0 0 0 0 0585 0 0920 0.146 0 1 03 0.118 0.894
Dehydronifedipine 0 E 0.00704 0 0 0 0 0 0 0 0 0 0 898 0.0940 0841
Caffeinet 3CISTD 1.12 117 1 14 1.44 1.33 126 1.19 1.34 1.15 1 17 122 1 1 1
Phenacetin13C 0 490000 0448 0 74B 0 935 0.673 0409 0619 0 629 0 499 0 535 115 102 00953 0784
* 1100% = sp/ke reccveiy
E = estimated (

Table 3.7.4. Sample set number 9285.05 results.
Lab ID Lab ID Lab ID Lab ID Lab ID Lab ID Lab ID Lab ID Lab ID Lab ID Set Blank Lab ID Set Spike Lab ID
Set Number Project Account Station ID Project Account Station ID Project Account Station ID Project Account Station ID Project Account Station ID Project Account Station ID Project Account Station ID Project Account Station ID Project Account Station ID Project Account Station ID ccv
Station Name Station Name Station Name Station Name Station Name Station Name Station Name Station Name Station Name Station Name
9285.05 991310006 488319401 07241550 North Canadian R Nr HarrahOK 991310007 488319101 07241560 North Canadian R Nt Harrah OK 991310019 489319401 06093500 8lue R Nr Kansas City, MO 991340098 468319401 07178200 Bud Cr at SH266 Nr Catoosa, OK 991340297 488319401 01100000 Merrimack R below Concord RV@ Lowell, MA 991380198 441711200 05525500 Sugar C at MiKcrd 991390234 488319401 06929315 Paddy Cr, Missouri 991400142 488319401 12000092 Indian-Moxlie Cr Outfall @ Olympia, WA 991410205 488319401 05531500 Sail Ci @ Western Sprigs, IL 991410206 488319401 05532500 Oes Plaines @ Rrrtrside, IL 92858040 92859040
Co'lecfion Date osmm 05433.99 059699 05/13799 05/12)99 05/1399 05/1599 05/1899 05/1999 05/1999
Collection Time 1045 1030 1130 1315 0640 1300 1315 1530 1400 1240
Compound Sample Volume Sample Cone Sample Volume Sample Cone Sample Volume Sample Cone Sample Volume Sample Cone Sample Volume Sample Cone Sample Volume Sample Cone Semple Volume Sample Cone Sample Volume Sample Cone Sample Volume Sample Cone Sample Volume Semple Cone Sample Cone Sample Cone* 0.100 ng/jtl 1.00 ng///l
(ml) M m (ml) IxW (ml) w (ml) W0 M W) (ml) W) (ml) wn M wo JnL. wo wo wo
903 40 90ft 97 807 97 696 46 7.35 34 81383 9196? Emm 746 68 699 77
Metformin 0 0 0 0 0 0 0 0 0 0 0 0 0 0070 0 892
Amoxicillin 0 0 0 0 0 0 0 0 0 0 0 0 00949 0956
Cotinine 0 0 E 00158 E 00157 E 0.0109 0 0 0 00478 E 0 0259 0 0837 00097 0967
Salbutamol 0 0 0 0 0 0 0 0 0 0 0 0620 0 0989 0929
Cimetidine 0 0 0 0 0 0 0 0 0 0 0 0297 0104 0 964
Acetaminophen E 8 70E-03 00112 E 6 60E-03 0 0.0403 0.0331 E 0 00500 0325 0.0257 0 E 0.0132 0.574 0115 0 963
Ranitidine 0 0 0 0 0 0 0 0 0 0 0 0 392 0099! 0 993
Paraxanthine 0 0 00968 0 0 0 0 0 0 0 0 0.873 0111 101
Lisinopril 0 0 0 0 0 0 0 0 0 0 0 0287 0 088 109
Enalaprilat 0 0 0 0 0 0 0 0 0 0 0 0112 0138 1 10
Trimethoprim 0 0 E 00109 0 E 000503 0 0 0 E 00132 E 00118 0 0782 0105 0941
Diqoxiqenin 0 0 0 0 0 0 0 0 0 0 0 0827 0103 0950
Paroxetine Metabolite 0 0 0 0 0 0 0 0 0 0 0 0 069 00904 0917
Oittiazem 0 0 0 0 0 0 0 0 0 0 0 0 799 0104 0960
Furosemide 0 0 0 0 0 0 0 0 0 0 0 0705 0130 1 06
Fluoxetine 0 0 0 0 0 0 0 0 0 0 0 0734 0102 0874
Warfarin 0 0 0 0 0 0 0 0 0 0 0 0860 0108 096?
Ibuprofen 0 0 0106 0 0 0 0 0 0 0 0 0799 0118 101
Gemfibrozil 0 0 00480 0 0 0 00303 0 0 0 0 0 736 0 099 100
Caffeine 0 0524 0 0750 0116 0.158 0.0062 0 0126 0217 0120 0 0 823 Oil 0950
Sulfamethoxazole 0 0 0 0 0 0 0 0 0 0 0 113 0119 0967
Dehydronifedipine 0 0 0 0 0 0 0 0 0 0 0 104 Oil 0969
Caffeinel 3CISTD 1.11 1 10000 124 144 136 1.23 109 115 1 34 143 120000 1 1 1
Phenacetinl 3C 128 130000 0 620 0.795 0765 0 803 0759 0739 0604 0435 1 11 0 959 0105 0864
* 1100%= spike reccwery
E = estimated (

Table 3.7.5. Sample set number 9285.06 results.
Set Number Lib ID Project Account Station ID Station Name Lib ID Project Account Station ID Station Namo Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Namo Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Namo Lab ID Project Account Station ID Station Namo Sot Blank Lab ID Set Spike Lab ID ccv
9285.06 992300189 451113600 335640084241901 Cobb County Waler Treatment Plant, GA 992380190 451313600 335640084252601 Cobb County Waler Intake. GA 992380131 451313600 335640064252601 Cobb County Water Intake, GA- Replicate 9973BJT52 1 451313600 334910084270001 AJlanta Water Works Chattahoochee Waler Treatment nu#.* r>* 992380193 45131)600 334939084271501 Allanla Waterworks Intake. GA 992420017 451313600 335837084713101 Johns Creek WPCP. GA-Field Blank 992420010 451313600 335837084713101 Johns Creek WPCP. GA Rep'icete 992420019 451313600 335732084153401 Crocked Creek WPCP. GA 992420020 451313600 335837004713101 Johns Creek WPCP. GA 992420033 451313COO 02335741 0tg Creek below Waler Works Intakes (City of Roswell). GA 992858041 992859041
Collection Date 08/25/99 10/25*9 08/25/99 08/25/79 08/25*9 08/27*9 08/27*9 08/27*9 08/27*9 C6/27*9
Collection Time 0945 1015 1020 1215 1200 1025 1020 0845 1015 1130
Compound Sample Volume M Semple Cone Wt Sample Volume M Sample Cone Wt Sample Volume (mi) Sample Cone wo Semple Volume M Semple Cone Wt Sample Volume (ml) Sample Cone wt Sample Volume (ml) Sample Cone wt Sample Volume (ml) Sample Cone wt Sample Volume (ml) Sample Cone wo Sample Volume (ml) Sample Cone wo Sample Volume (ml) Sample Cone wt Sample Cone wt Sample Cone wt 0.100 1.00 n^l
870 11 834 09 86607 884 63 884 86 8916 89232 904 83 9108 890 94
Metformin 0 0 0 0 0 0 0 0 0 0 0 0.00843 0108 0943
Amoxicillin 0 0 0 0 0 0 0 0 0 0 0 0 000 0107 1.01
Cotinine 0 E 00132 E 00130 E 00135 0 0 E 00164 0 0 E 00140 0 0775 0105 1 12
Salbutamol 0 0 0 0 0 0 0 0 0 0 0 0582 0116 0 930
Cimetidine 0 0 0 0 0 0 0 0871 0 0 0 0311 0111 0967
Acetaminophen 00113 00126 00138 0 00135 00124 0 0 0 E 0 00820 0.0145 0581 0.123 0906
Ranitidine 0 0 0 0 0 0 0 0 0 0 0 0403 0109 0964
Paraxanthine 0 00282 0 0 0 0 0 0 0 0 E 0 00700 0 940 0107 1 Ofi
Lisinopril 0 0 0 0 0 0 0 0 0 0 0 0315 013 1.12
Enalaprilat 0 0 0 0 0 0 0 0 0 0 0 0151 0.111 1.05
Trimethoprim E 000336 0 0 0 0 0 0 08/4 E 00120 00783 0 E 0 00100 0787 0111 0 931
Digoxiqenin 0 0 0 0 0 0 0 - 0 0 0 0 0872 0118 0937
Paroxetine Metabolite 0 0 0 0 0 0 0 0 0 0 0 0102 0102 100
Diltiazem 0 0 0 0 0 0 0 0 0485 0 0 E 000890 0775 0108 0935
Furosemide 0 0 0 0 0 0 0 0 0 0 0 0667 0115 0 938
Fluoxetine 0 0 0 0 0 0 0 0 0 0 0 0 591 0.106 0938
Warfarin 0 0 0 0 0 0 0 0 0 0 0 0618 0111 0936
Ibuprofen 0 0 0 0 0 0 0 0 0 0 0 0772 0 0958 1.01
Gemfibrozil 0 0 0 0 0 0 0 0 0 0 0 0757 0113 0940
Caffeine 0 0417 0 0765 00764 00572 0 0758 E 00122 0 00190 0 00489 0 0 826 0104 0920
Sulfamethoxazole 0 0 0 0 E 00183 0 0390 123 0453 0 0 110 0112 0 93)
Dehydronifedipine 0 0 0 0 0 0 00159 00207 00152 0 0 104 0.106 0945
Caffeinel 3CISTD 115 120000 1.15 113 1.13 1.12 112 1.10 1.10 112 114 1 1 1
Phenacetinl 3C 0 662 0 569 0513 0630 0 506 1 14 0546 0 544 0544 0 401 102 0 984 0111 0908
1100% = spike fecoverjc
£ = estimated (

Table 3.7.6. Sample set number 9287.04 results.
Set Number Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Set Blank Lab ID Set Spike Lab ID CCV
9287.04 991410207 4B8319401 06536996 C$SC@Romecriille H 991440075 488319401 03274000 Great Miami @ Hamilton, OH 991480291 488319401 02338000 Chatlahooche R Nr Whrlesburg. GA 991520248 488319401 07325860 Willow Creek Nr Albert 99152Q306 488319401 351000097264001 Norman Wastewater Outfall 991530198 488319401 01381600 Whippany R Nr Pine Brook, NJ 991530199 488319401 01391000 HoHoKus Brook at HoHoKus.MJ 991540124 488319401 04200504 Cuyahoga R at LTV Steel 991620033 488319401 365325092265801 Columbia Wetlands Units 1 991660215 488319401 10171000 Jordan Rwer at 1700 South 992878040 992879040
Collection Date 05/2099 05/20/99 05/24/99 mm 05/27/99 05/27/99 05/27/99 mm 06433/99 06/144)9
Collection Time 0810 1000 1420 1300 1053 1550 1120 1145 1300 1140
Compound Sample Volume (mO Sample Cone wo Sample Volume (ml) Sample Cone wo Sample Volume (ml) Sample Cone wo Sample Volume (ml) Sample Cone wo Sample Volume (ml) Sample Cone wo Sample Volume (ml) Sample Cone wo Sample Volume (ml) Sample Cone ten Sample Volume (ml) Sample Cone wo Sample Volume (ml) Semple Cone wt Sample Volume (ml) Sample Cone wt Sample Cone wo Sample Cone* wo 0.100 ng//il 1.00 ng/jd
62912 76150 89143 063.45 649 15 599 45 768 00 726 07 96306 863 98
Metformin E 0.135 E 00418 0 0 0 0 E 0 0795 E 0153 0 0 0 000345 0.109 0.997
Amoxicillin 0 0 0 0 0 0 0 0 0 0 0 0 000 0044 0148
Cotinine 0 f 0 0244 0 0 0 E 00188 00387 0 0502 0 E 00174 0 0 888 0 0981 101
Salbulamol 0 0 0 0 0 0 0 0 E 00101 0 0 0 778 0.0881 0936
Cimetidine 0.0534 0 0 0 1.68 0 00964 0 3.53 0 0 0 382 0.115 1.00
Acetaminophen 0 0 0 E 0 00283 0 0 0 0 0 0 0 0550 0.109 096
Ranitidine 0 0 0 0 0134 0 0 0 0 . 0 0 0433 00652 0994
Paraxanthine 0 0 0 0 00966 0 0 0 00652 0 0 0723 00955 0968
Lisinopril 0 0 0 0 0 0 0 0 0 0 0 0224 0 127
Enalaprilat 0 0 0 0 0 0 0 0 0 0 0 0.0569 0.101 0874
Trimethoprim 0 0596 E 0 00975 00361 0 0 302 0 00776 0 00375 0 0 0 873 0 0988 0898
Digoxigenin 0 0 0 0 0 0 0 0 0 0 0 0845 0 098 0 886
Paroxetine Metabolite 0 0 0 0 0 0 0 0 0 0 0 0 253 0256 1.58
Diltiazem 00232 0 00136 E 0.0107 00414 0 0.0214 0 0264 0 0 0 0822 0.106 1.01
Furosemide 0 0 0 0 0 0 0 0 0 0 0 0754 0 0 889
Fluoxetine 0 0 0 0 0 0 0 0 0 0 0 0 687 0105 0 999
Warfarin 0 0 0 0 0 0 0 0 0 0 0 0814 0 0909 0875
Ibuprofen 0 0 0 0 0 0 0 0 0 0 0 0024 0 0965 1 04
Gemfibrozil 0 0 0 0 0 0 0 0 0556 0 0 0.585 00974 108
Caffeine 00562 E 000603 E 000113 0 0102 E 0.0129 E 00146 0 0114 0 0 0 896 0 0855 0 991
Sulfamethoxazole 00392 0 0.16 0 0946 0 0 0 102 £ 00227 0 0 936 0106 0 685
Dehydronifedipine E 00111 0 E 00116 0 0 E 00132 00196 00185 00131 0 0 1069 0108 103
Caffeine! 3CISTD 1.12 131 1.12 113 1.18 1.67 13 1.38 104 1.16 1.11 1 1 1
Phenacetinl 3C 0 392 0620 0593 0.514 0411 0 608 0506 0.497 0308 0.472 1.11 0 959 0.104 0 958
1100%= spike recovery
E = estimated (

Table 3.7.7. Sample set number 9287.06 results.
Set Number Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Set Blank Lab ID Set Spike Lab ID ccv
9287.06 451313600 02335640 Kelly Mill Branch Tributary al Castleberry Road. r * 992020145 451313600 341145064111201 City of Cummings WPCP.GA 992020190 451313600 334939064271501 Atlanta Waler Works Intake, GA 992020192 451313600 02335741 Big Creek below Waterworks Intakes (Cily of Roswell), GA 992020193 451313600 340108084210801 City of Roswell Water Treatment Plant, GA 451313600 33491008427080! Ajianta Waterworks Chattahoochee Water Treatment 397rorr 451313600 334918084270801 Atlanta water Works- Chattahoochee Water Treatment 992110175 488319401 08155240 Lost Creek Spring, IX 992220141 488319401 08057410 Trinity R below Dallas, IX 992380188 451313600 340003084232401 Big Creek WPCP. GA 992878041 992679041
Collection Dale 07/1 m 07/19,99 07/19/59 07/19/59 07/19/99 07/2CW9 07/2093 07/29/99 08439/39 08/25/99
Collection Time 1600 1416 1000 1000 1215 1300 1100 0900 0830 0845
Compound Semple Volume M Sample Cone Sample Volume M Sample Cone wo Sample Volume m Sample Cone M Sample Volume M Sample Cone WO Sample Volume H Sample Cone W Sample Volume (ml) Sample Cone wo Sample Volume (ml) Sample Cone wo Sample Volume (ml) Sample Cone wo Sample Volume (ml) Sample Cone wo Sample Volume (mf) Sample Cone wo Sample Cone wo Sample Cone W) 0.100 ng/pl 1.00 ng/pl
840 96 826 28 65631 073 13 717 12 89959 877 51 906 76 84145 9126
Metformin 0 E 0215 0 0 0 0 0 0 E 0 0474 E 0142 0 00123 00999 095
Amoxicillin 0 0 0 0 0 0 0 0 0 0 0 0 000 00789 0 935
Cotinine 00791 E 00205 0 0 0 E 00142 0 0 00593 E 00165 0 0.749 0.11 108
Salbutamol 0 0 0 0 0 0 0 0 0 0 0 0 734 0.118 0966
Cimeddine 0 0 0 0 0 0 0 0 0 0 664 0 0 416 0.115 106
Acetaminophen 0 0 0 C 6 10E-O3 E 8.60E-O3 E 000397 0 00111 0 0 0 0 599 0108 0.934
Ranitidine 0 0 0 0 0 0 0 0 0 0 0 0465 0.111 1 14
Paraxanthine 0 0 E 00192 0 0 0 0 0 0 0 0 0 821 00896 0928
Lisinopril 0 0 0 0 0 0 0 0 0 0 0 0187 00311 12
Enalaprilat 0 0 0 0 0 0 0 0 0 0 0 00713 0 0929 0 837
Trimethoprim 0 0 0 0 0 0 0 0 0 00644 0 0 833 0121 105
Digoxigenin 0 0 0 0 0 0 0 0 0 0 0 0 822 0.113 0 994
Paroxetine Metabolite 0 0 0 0 0 0 0 0 0 0 0 0 609 0 0896 1.5
Diltiazem 0 0 0 0 0 0 0 0 0 0.0517 0 0865 0104 102
Furosemide 0 0 0 0 0 0 0 0 0 0 0 0 740 0152 104
Fluoxetine 0 0 0 0 0 0 0 0 0 0 0 0 669 0105 0 966
Warfarin 0 0 0 0 0 0 0 0 0 0 0 0 832 0092 0 893
Ibuprofen 0 0 0 0 0 0 0 0 0 0 0 0 773 0.105 108
Gemfibrozil 0 0 0 0 0 0 0 0 0 0 0 0 631 00818 0 996
Caffeine 0.786 00175 0 0459 0 00223 0 0227 0 0 00503 E 00111 0 0976 00927 0992
Sulfamethoxazole 0 0 E 00200 0 0 0 0 E 0.0106 0 0573 0 0 935 0113 0944
Dehydronifedipine 0 0 0 0 0 0 0 0 00274 00190000 0 1.09 0114 108
Caffeinel 3CISTD 1.19 1.21 1.16 1.14 139 111 114 1.10 1 19 1.10000 1.16 1 1 1
Phenacetinl 3C 0701 0 601 0534 0993 0 482 0471 0654 0740 0435 0 502 112 102 0104 0905
* x 100% = spike recwery
E estimated (

Table 3.7.8. Sample set number 9292.03 results.
MRDP Schedule 9003
Sample Set Report
Set Number Lab ID Project Account Station ID Station Namo Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Lab ID Project Account Station ID Station Name Set Blank Lab ID Set Spfke Lab ID CCV
9292.03 991690216 488319401 04127490 Boaidman R Nr Traverse Cily 991960106 462714102 05331580 Mississippi R bSv LD2 al Hastings, MN 992020111 451313600 335837084173101 Johns Creek WPCP. GA 992020112 451313600 335732084153401 Crooked Creek WPCP.GA 992020113 451313600 335640084241901 Cobb County Water Treatment Plant, GA 992020114 451313600 335640084252601 Cobb County Water Intake, GA 992020115 451313600 340333084232401 Big Creek WPCP. GA 992929040 992929040
Collection Date 06/1609 07/13-99 07/1609 07/1609 07/16/99 07/1609 07/1609
Collection Time 1045 1300 1045 0830 1430 1500 1300
Compound Sample Volume (ml) Sample Cone two Sample Volume (ml) Sample Cone M Sample Volume (ml) Sample Cone wo Sample Volume (ml) Sample Cone (W# Sample Volume (ml) Sample Cone Sample Volume (ml) Sample Cone wo Sample Volume H Sample Cone wo Sample Volume (ml) Sample Cone wo Sample Volume (ml) Sample Cone wo Sample Volume (mi) Sample Cone wo Sample Cone wo Sample Cone' wo 0.100 ng/)il 1.00
046 71 674 57 61901 901.11 864 25 761.57 623.92
Metformin 0 0 0 0 0 c 0 0 000481 0112 1 12
Amoxicillin 0 0 0 0 0 0 0 0 000 00545 0173
Cotinine 0 0 OQ572 0 00165 0 0 0 0 868 00921 1 04
Salbutamol 0 0 0 0 0 0 0 0 0.725 0.115 103
Cimetidine 0.0116 0 0 0252 0 0 1.88 0 0410 0124 108
Acetaminophen l 7 70E-03 0 0 0 E 7 90EQ3 00142 0 E 0.00570 0 570 0103 0903
Ranitidine 0 0 0 0 0 0 0 0 0492 0 0678 1.01
Paraxanthine 0 0 0.226 0 0 00420 0 0 0708 00966 0971
Lisinoprif 0 0 0 0 0 0 0 0 0290 00835 132
Enalaprilat 0 0 0 0 0 0 0 0 0103 00899 0848
Trimethoprim E 00112 0 0.109 0 0 E 6 90E-03 0 0486 0 0.824 00976 0913
Diqoxiqenin 0 0 0 0 0 0 0 0 0 852 0 0992 0884
Paroxetine Metabolite 0 0 0 0 0 0 0 0 0 204 0196 1 55
Diltiazem 0 0 0 0.0332 0 0 00500 0 0873 0105 104
Furosemide 0 0 0 0 0 0 0 0 0775 0126 1 09
Fluoxetine 0 0 0 0 0 0 0 0 0 752 0106 104
Warfarin 0 0 0 0 0 0 0 0 0870 00937 0 906
Ibuprofen 0 0 0 0 0 0 0 0 0005 00941 102
Gemfibrozil 0 0 0921 0 0 0 0 0 0710 0094 1 10
Caffeine 0 0 106 E 00135 0166 0 254 00260 0 0947 0.0914 0931
Sulfamethoxazole 0 0 0134 0.911 0 E 0 0236 0 369 0 1.00 0111 0958
Dehydronifedipine 0 0 0 00194 0 0 00222 0 1 09 0104 106
Caffeine! 3CISTD 1 16 1 46 1 22 1.11 1 17 1 31 121 1 12 1 1 1
Phenacetin13C 0.594 0.506 0.438 0.671 0844 0.000957 0639 106 0998 0103 0 951
* 100% = spike recovery
E = estimated (

Table 3.7.9. Sample result summary.
Compound Mean all samples jig/l (n=77) Mean, hits only, ^g/l Median hits only pg/1 n hits Max cone, pg/l Min cone, pg/l
Metformin 0.0109 0.120 0.139 8 0.215 0.0418
Amoxicillin 0.00 0.00 0.00 0 0.00 0.00
Cotinine 0.0249 0.0610 0.0175 31 0.896 0.0109
Salbutamol 0.00 0.00 0.00 0 0.00 0.00
Cimetidine 0.137 0.803 0.604 15 3.53 0.0116
Acetaminophen 0.146 0.370 0.0112 25 10.0 0.000500
Ranitidine 0.00 0.00 0.00 0 0.00 0.00
Paraxanthine 0.0372 0.236 0.0727 12 1.90 0.0192
Lisinopril 0.00 0.00 0.00 0 0.00 ' 0.00
Enaiaprilat 0.00 0.00 0.00 0 0.00 0.00
Trimethoprim 0.0205 0.0553 0.0301 27 0.303 0.00336
Digoxigenin 0.00 0.00 0.00 0 0.00 0.00
Paroxetine Metabolite 0.00 0.00 0.00 0 0.00 0.00
Diltiazem 0.00663 0.0294 0.0264 16 0.0612 0.00243
Furosemide 0.00 0.00 0.00 0 0.00 0.00
Fluoxetine 0.00 0.00 0.00 0 0.00 0.00
Warfarin 0.00 0.00 0.00 0 0.00 0.00
Ibuprofen 0.0165 0.417 0 108 3 1.05 0.0941
Gemfibrozil 0.0322 0.490 0.556 5 0.921 0.0480
Caffeine 0.173 0.248 0.0758 53 6.04 0.00113
Sulfamethoxazole 0.105 0.362 0.193 22 1.23 0.00905
Dehydronifedipine 0.00347 0.0164 0.0172 14 0.0295 0.00480
Phenacetin13C 0.600 0.600 0.556 77 1.30 0.297
74


Table 3.7.10. Spike recovery summary.
Spike Recovery (n=8) Average Spike Reccvetv Standard deviation %Stddev
Metformin 0.00 0.00 0.00908 0.00 0.00843 0.00945 0.0123 0.00481 0.00551 0.00499 90.6
Amoxicillin 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Cotinine 0.761 0.667 0.723 0.837 0.775 0.888 0.749 0.868 0.784 0.0754 9.62
Salbutamol 0.754 0.584 0.659 0.620 0.582 0.778 0.734 0.725 0.679 0.0784 11.5
Q'metidine 0.305 0.312 0.258 0.297 0.311 0.382 0.416 0.410 0.336 0.0582 17.3
Acetaminophen 0.682 0.553 0.761 0.574 0.581 0.550 0.599 0.570 0.610 0.0758 124
Ranitidine 0.387 0.286 0.353 0.392 0.403 0.433 0.465 0.492 0.401 0.0647 16.1
Paraxanthine 0.995 0.799 0.999 0.873 0.940 0.723 0.821 0.788 0.867 0.102 11.8
Lisinopril 0.441 0.318 0.446 0.287 0.315 0.224 0.187 0.290 0.313 0.0919 29.3
Enalaprilat 0.125 0.110 0.172 0.112 0.151 0.0569 0.0713 0.103 0.113 0.0380 33.7
Trimethoprim 0.846 0.749 0.785 0.782 0.787 0.873 0.833 0.824 0.810 0.0408 5.04
Qgoxigenin 0.863 0.793 0.911 0.827 0.872 0.845 0.822 0.852 0.848 0.0356 4.20
Paroxetine Metabolite 0.0943 0.00 0.0252 0.069 0.102 0.253 0.609 0.204 0.169 0.197 116
Dltiazem 0.733 0.701 0.677 0.799 0.775 0.822 0.865 0.873 0.781 0.0727 9.32
Furosenide 0.648 0.527 0.629 0.705 0.667 0.754 0.740 0.775 0.680 0.0808 11.9
Fluoxetine 0.932 0.759 0.842 0.734 0.591 0.687 0.669 0.752 0.746 0.105 14.1
Warfarin 0.890 0.851 0.867 0.860 0.818 0.814 0.832 0.870 0.850 0.0269 3.16
Ibuprofen 0.754 0.719 0.789 0.799 0.772 0.824 0.773 0.805 0.779 0.0327 4.20
Gemfibrozil 0.766 0.644 0.820 0.736 0.757 0.585 0.631 0.710 0.706 0.0794 11.2
Caffeine 0.965 0.828 0.888 0.823 0.826 0.896 0.976 0.947 0.893 0.0639 7.15
Sulfamethoxazole 0.957 0.815 1.03 1.13 1.10 0.936 0.935 1.00 0.988 0.101 10.2
Dehydronifedpine 0.962 0.902 0.898 1.04 1.04 1.069 1.09 1.09 1.01 0.0798 7.89
Phenacetin13C 1.094 0.953 1.02 0.959 0.984 0.959 1.02 0.998 0.999 0.0472 4.73
4. Discussion
4.1 Target Analytes
The total mass determined for each compound was the primary factor in developing a
target analyte list. Figures for total production of drug compounds are not available in the
literature. Thorough breakdowns of pharmaceutical sales as determined by commercial market
tracking are proprietary and costly. The masses derived for the compounds listed in table 3.1.1
are most likely underestimated as there are usually numerous brands and dosages of each brand
that are dispensed. The drug amoxicillin can be related to fifteen headings in the Physicians
75


Desk Reference, among those headings a number of forms including liquid, tablet, and capsule,
all at multiple dosages (PDR 1998).
Of the top 20 drugs prescribed as listed and shown in table 3.1.1, a number of drugs are
prescribed at very low dosage. The unit doses for levothyroxin, conjugated estrogens,
salbutamol, and digoxin are less than 1 mg. Even at very high numbers of prescriptions the
combined total mass determined for the two brands of conjugated estrogens was less than 1000
kg. The conjugated estrogens contain a mixture of six sodium salts of estrogen sulfates
representing the average composition of estrogens in pregnant mares urine (RxList). Therefore
the mass of any one of the estrogen sulfates is only a fraction of the total mass prescribed.
Synthroid (levothyroxin), a common hypothyroid medication was only determined to be prescribed
in total mass of 104 kg. Levothyroxin appears under fewer headings in the literature than does
albuterol (salbutamol) and has a consistently low dosage. Salbutamol appears under numerous
headings and with different forms and dosages up to 4mg. It is likely to be underestimated in
total mass to a greater extent than the other low dose medications. In addition SPE recovery (Fig.
3.5.1.4) and the mass spectral response (Fig. 3.2.2) of salbutamol under experimental conditions
was very good. Therefore, salbutamol was selected on the basis that it is present but in relatively
small quantity and that the ability or inability to find such a compound in surface water would
provide valuable information for the study. Likewise, digoxin is of relatively low abundance but it
is also found in nature as a product of the foxglove plant. It also responded well to SPE and LC-
MS (Figs. 3.2.2, and 3.5.1.4). Therefore curiosity rather than likelihood of discovery supported
the decision to include this compound in the work. The remainder of the compounds listed in
table 3.1.3 was selected based upon high mass numbers and availability. Some compounds
such as norsertraline or norfluoxetine (Table 3.1.2) were either difficult to obtain or prohibitively
expensive.
76


A determination of the form of the drug (parent compound or metabolite) to analyze for
had to be made for each compound selected. Some drugs are excreted unchanged and others
are biotransformed. Biotransformation is divided into two categories: metabolism and
conjugation. Metabolic reactions include oxidation, reduction, and hydrolysis. Conjugation
reactions combine products of metabolism with endogenous constituents such as amino acids,
glucuronic acid, and sulfate (Liu and Gadzala 1997). However, metabolites formed by
conjugation such as glucuronides and sulfides are likely to be cleaved in the environment into the
original or unconjugated form (Ternes 1998). Table 4.1.1 lists the analytes from table 3.1.3
together with the forms that are excreted with percentages of the dose if known. In addition, the
reason the analyte was chosen is briefly described.
77


Table 4.1.1. Determination of target analytes from biotransformation references.
Drug name Form Eliminated, % of dose Form chosen for study Reason Reference(s)
Amoxicillin Unchanged, 60% Amoxicillin Main form (RxList)
Metformin Unchanged, 90% Metformin Main form (RxList)
Cimetidine Sulfate, 48% Cimetidine Cleavage, hard to obtain metab. (RxList)
Gemfibrozil Glucuronide, 32% Gemfibrozil Cleavage, hard to obtain metab. (Nakagawa, Shigeta et al. 1991; Thomas, Burgess et al. 1999)
Sulfamethoxazole N4-Acetyl, 48-60% Unchanged, 14-30% Sulfamethoxazole Flard to obtain metab. (RxList; Vree, Ven et al. 1995)
Diltiazem Desacetyldiltiazem Diltiazem Could not obtain metab. (RxList; Clozel, Caille et al. 1984; Flussain, Tam et al. 1992)
Ranitidine Unchanged 30% Ranitidine Main form (RxList)
Furosemide Unchanged, Glucuronide Furosemide 3 to 1 ratio to metab. (Russel, Tan et al. 1989; Abou-Auda, Al-Yamani et al. 1998)
Nifedipine Dehydronifedipine, 80% Dehydronifedipine Main form (RxList; Tsuruta, Nakamura et al. 1997; Streel, Zimmer et al. 1998)
Trimethoprim Unchanged, 80% Trimethoprim Main form (RxList; Mengelers, Polman et al. 1993)
Paroxetine BRL36610, 64% Glucuronide, sulfate BRL 36610 Main form (RxList; Harder, Hermes et al. 1994; Foglia, Sorisio et al. 1997)
Fluoxetine Norfluoxetine, 50% Unchanged, 10% Fluoxetine Could not obtain metab. (RxList; Crifasi, Le et al. 1997)


Table 4.1.1 cont.
Drug name Form Eliminated, % Form chosen for study Reason Reference(s)
of dose
Lisinopril Unchanged, 6-60% Lisinopril Main form (RxList)
Enalapril Enalaprilat, 40% Unchanged, 20% Enalaprilat Main form (RxList; Shioya, Shimojo et al. 1992)
Digoxin Unchanged, 50-80% Digoxigenin Cleavage of digitoxose units (RxList; Eriksson, Tekenbergs et al. 1981)
Warfarin Unchanged, 92% Warfarin Main form (RxList; Spink, Aldous et al. 1989)
Salbutamol Sulfate, 60% Salbutamol Hard to obtain metab. (RxList; Boulton and Fawcett 1995; Walle, Eaton et al. 1996; Logsdon, Zhou et al. 1997)
Acetaminophen Glucuronide Acetaminophen Cleavage, hard to obtain metab. (Colin and Sirois 1986; Goicoechea, Lopez de Alda et al. 1995)
Caffeine 1,7-Dimethylxanthine, 12 other forms 1,7-Dimethylxanthine, Caffeine Main form, Disposal of drinks (Liguori, Mascara et al. 1991; Krul and Hageman 1998)
Ibuprofen Glucuronide Ibuprofen Cleavage, hard to obtain metab. (RxList; Tan, Hutt et al. 1995; Kepp, Sidelmann et al. 1997; Tan, Jackson et al. 1997)
Nicotine Nicotine, 10% Cotinine, 15% trans- Hydroxycotinine, 45% Cotinine Hard to obtain THOC (RxList; Rustemeier, Demetriou et al. 1993)


4.2 High Performance Liquid Chromatography
High-performance liquid chromatography was developed using 10mM ammonium
formate buffer and acetonitrile in the mobile phase. The ammonium formate buffer at pH 3.7
has the ability to protonate all the compounds of interest, pka>4, and has high volatility which
enhances production of ions in the electrospray source (Snyder, Kirkland et al. 1997).
Acetonitrile was used as the organic solvent in the mobile phase. It has higher solvent
strength at lower concentrations than methanol and is more capable of resolving the
compounds that elute from the column at higher organic concentrations (Snyder, Kirkland et
al. 1997), which is where the majority of the compounds were eluting. The base deactivated
column also yielded narrower peaks in the early eluting, more polar compounds compared to
a regular end-capped C-18 column.
A stepped gradient (Table 3.2.2) was required to optimally resolve the compounds.
Coelution was not a real concern because SIM and ion extracting software was used.
However, minimizing the number of compounds eluting within one SIM low was of
importance. Fewer ions in a SIM window means more dwell per ion by the analyzer, hence
more sensitivity. The chromatogram in figure 3.2.1 shows the initial condition with a large
number of coelutions. Figure 3.2.2 shows peaks resolved from what would have been two to
four SIM windows at the beginning of the chromatogram. Starting and holding at a low
concentration of 6% acetonitrile allowed sufficient time for the compounds to interact with the
column. Then a gentle gradient was used to separate the more polar compounds. For the
later eluting, less polar compounds, a quick step up to a higher organic concentration then a
slow gradient made for acceptable resolution.
4.3 Mass Spectral Characterization of Compounds
Mass spectra of the compounds were studied by using flow injection analysis and
varying the fragmentor (capillary exit) voltage in the mass spectrometer. The major definitive
80


ions for each compound are listed in table 3.3.1. In all but one case the (M+H)+ ion was a
major ion in mass spectra. A sodium adduct resulted for furosemide (M+Na)+ at all
fragmentor settings, with only one other discernable consistent fragment. All structures were
examined to account for fragmentation and where possible referenced for corroboration. The
following figures represent interpretation of fragmentation information from mass spectra.
VSj' 3. a.Tie-6S.53u 01 . 'WIX5- D AR-ES Ffcsisvo j
100-j a>S 30040 1
80- i i
60- I j
40- i ;
20- 85 0 88.1 113 0
1 . 1 i s
62 80 m 12s ! id
(M+H)* 130
113
NH NH
NH,
Figure 4.3.1. Metformin, formula wt. 129, fragment 113, (M+H)+ 130, fragmentor 80V.
Figure 4.3.2. Amoxicillin, formula wt. 365, fragments 114*, (208 not interpreted), 349,
fragmentor 70V (Straub and Voyksner 1993).
VC, oi c 4 VIX AtVsib
Max.
(M+H)+177
Figure 4.3.3. Cotinine, formula wt. 176, fragments 98, 80, (M+H)~ 177, fragmentor 90V.
81


Figure 4.3.4. Salbutamol, formula wt. 239, fragments 222, 166*, (M+H)+ 240, fragmentor
70V.
(M+H)+ 253
Figure 4.3.5. Cimetidine, formula wt. 252, fragments 159, (117 not interpreted), (M+H)*253
fragmentor 80V.
Figure 4.3.6. Acetaminophen, formula wt.151, fragments 110*, 93, (M+H)+ 152, fragmentor
87V.
82


| ' $PC. .ne=3fl of *\FW24X C AP-S-S As.lr.-e
]
100-i
50 J,
Max:^7C62<
(M+H)+315
176
H
'Nv H
-N
20-i
\
*'0
_!BL
_22£_________nii
Figure 4.3.7. Ranitidine, formula wt. 314, fragments 270, 176, (M+H)+315, fragmentor 80V.
(M+Hp81
*MSD1 SPC wnea103.927ofF.DR!jGLO.B\MK54.MB<54.C APVES Positive
Sax- 27016 I
60 H
J
1
20-j
Figure 4.3.8. 1,7-Dimethylxanthine, formula wt.180, fragment 124, (M+H)+181, fragmentor
Figure 4.3.9. Lisinopril, formula wt. 405, fragment 246*, (M+H)+406, fragmentor 90V.
83


Figure 4.3.10. Caffeine, formula wt. 194, fragments 138, 110*, (M+H)+195, fragmentor
100V.
Figure 4.3.11. Enalaprilat, formula wt. 348, fragments 303, 206*, (M+H)+349, fragmentor
100V.
(M+H)+291
*MS01 SPC. time =92.322 Of F \DRUGLCJ5'.MiX54\M!X54 b AFVES Pos4ive
Figure 4.3.12. Trimethoprim, formula wt. 290, fragments 261*, 230, (M+Hf 291, fragmentor
120V.
84


(M+H)+391
Figure 4.3.13. Digoxigenin, formula wt, 390, fragments 373, 355, (M+H)+391, fragmentor
100V.
F
(M+H)+332
Figure 4.3.14. Paroxetine metabolite, formula wt 331, fragments 192, 109*, (M+H)+332,
fragmentor 120V.
Figure 4.3.15. Diltiazem, formula wt. 414, fragments 370, 178*, (M+H)+415, fragmentor
105V.
85


Figure 4.3.16. Furosemide, formula wt. 330, fragment 81, (M+Na)+ 353, fragmentor 120V.
Figure 4.3.17. Ibuprofen, formula wt. 206, fragment 161, (M+H)+ 207, fragmentor 60V.
(M+H)+309
Figure 4.3.18. Warfarin, formula wt. 308, fragments 163, 251, (M+H)+ 309, fragmentor 70V.
86