AN ABSTRACT OF THE THESIS OF
Yan Hu for the degree of Master in Science in Chemistry presented on June 19, 2009.
Title: Determination of Ethyl Glucuronide and Ethyl Sulfate as Human Biomarkers of
Alcohol Exposure in Urine by Liquid Chromatography/Electrospray Tandem Mass
Spectrometry with Large Volume Injection
Abstract approved:
_____________________________________________________________________
Jennifer A. Field
Staci L. Simonich
This research was undertaken to develop a sensitive, selective, and rapid analytical
method to measure ethyl glucuronide (EtG) and ethyl sulfate (EtS) in urine as urinary
biomarkers of alcohol exposure. Large-volume injection (LVI) coupled with liquid
chromatography / electrospray tandem mass spectrometry was used to eliminate the
need for either off-line or on-line solid phase extraction. Urine sample preparation
was limited to diluting the urine 1:1000 with deionized water prior to large-volume
injection (900 μL). Three MS/MS transitions were monitored for EtG and two
MS/MS transitions were monitored for EtS, with the deprotonated molecular ion
[M-H]- as the precursor ion: m/z 221→75, 221→85, 221→113(EtG) and 125→80,
125→97(EtS). Pentadeuterated EtG and pentadeuterated EtS were used as internal
standards for EtG and EtS, respectively. The method was accurate with recoveries
from 99.8% to 102.9% for EtG and 93.3% to 102.5% for EtS and gave good
precision, as indicated by average RSD, of 3.5% and 4.9% for EtG and EtS,
respectively. Matrix effects were eliminated and the instrumental detection limit was
0.005 μg/mL for EtG and 0.010 μg/mL for EtS, which is a factor of 10 lower than
previous published analytical methods for measuring EtG and EtS. The method
quantification limits were 0.015 μg/mL for EtG and 0.025 μg/mL for EtS in 1:1000
diluted urine, corresponding to 15 μg/mL for EtG and 25 μg/mL for EtS in undiluted
urine. Finally, the developed method gave statistically equivalent results when
applied to measuring EtG and EtS in a third-party reference urine.
©Copyright by Yan Hu
June 19, 2009
All Rights Reserved
Determination of Ethyl Glucuronide and Ethyl Sulfate as Human Biomarkers of
Alcohol Exposure in Urine by Liquid Chromatography/Electrospray Tandem Mass
Spectrometry with Large Volume Injection
by
Yan Hu
A THESIS
Submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Presented June 19, 2009
Commencement June 2010
Master of Science thesis of Yan Hu presented on June 19, 2009.
APPROVED:
Co-major Professor, representing Chemistry
Co-major Professor, representing Chemistry
Chair of the Department of Chemistry
Dean of the Graduate School
I understand that my thesis will become part of the permanent collection of Oregon
State University libraries. My signature below authorizes release of my thesis to any
reader upon request.
Yan Hu, Author
ACKNOWLEDGEMENTS
I would like to give special thanks to my advisors Dr. Staci Simonich and Dr. Jennifer
Field for their guidance, support and input throughout the course of my studies. Many
thanks to my academic committee: Dr. Claudia Maier, and Dr. Fredrick Prahl, for
their advice on the dissertation content throughout the committee meetings and
examinations.
Thanks also to Aurea Chiaia, Dr. Jeff Moore and the Simonich lab for their expertise.
Thanks to the faculty and staff of the Department of Chemistry and Department of
Environmental and Toxicology who directly or indirectly contribute to my work.
Warmly thanks to my friends for their support and good times.
Finally, I would like to thank my parents for all their love, support and
encouragement throughout the development of my thesis.
TABLE OF CONTENTS
Page
1
Introduction……………………………………………………………………..1
2
Experimental …………………………………………………………………...11
2.1
Standards and Reagents………………………………….………………..11
2.2
Preparation of Stock and Working Solutions………….…...…………....12
2.3
Sample Preparation………………………………………..………...….....13
2.4
Instrumentation…………………………………………….………….…..14
2.4.1 High Pressure Liquid Chromatography (HPLC)………….….………14
2.4.2 Electrospray Tandem Mass Spectrometry(ESI-MS/MS)……...…......14
3
2.5
Injection Volume and Dilution Factor Optimization…...……………........19
2.6
Specificity…………………………………………………………………20
2.7
Stability…………………………………………………………………...20
2.8
Matrix effects…………………………………………...…………………21
2.9
Validation ………………………………………………………………...21
Results and Discussion ………………………………………………………22
3.1
Method Development…………………………………………..…………22
3.1.1 Chromatography………………………………………………….…..23
3.1.2 Optimization of Mobile Phase……………………………………..…25
3.1.3 Optimization of Injection Volume……………………….…………..28
3.1.4 Optimization of Sample Dilution Factor………………….........….29
TABLE OF CONTENTS (Continued)
Page
3.2
Method Validation…………………………………………………...……30
3.2.1 Specificity………………………………………………………….…30
3.2.2 Linearity………………………………………………………...……32
3.2.3 Matrix Effects……………………………………………….……….34
3.2.4 Accuracy and Precision……………………………………………..36
3.2.5 Detection and Quantification Limits…………………………………39
3.3
Method Verification………………………..…………………......………39
4
Conclusions………………………………………………………...……………41
5
Acknowledgements…………………………………………………………...…42
6
References…………………………………………………………………...…..43
LIST OF FIGURES
Figures
Page
1. Elimination pathways for ethanol in the human body, showing the major,
oxidate route, and the minor conjugation reactions with sulfate and
glucuronic acid……………………………………………………….....……..…..2
2. Chemical structures and molecular formulas of EtG and EtS and pentadeuterated internal standards……………………………………………………....4
3. Product-ion spectra of EtG and EtG-D5…………………………………………18
4. Product-ion spectra of EtS and EtS-D5 ……….………………………………...18
5. Modified injection of an Agilent 1100 HPLC with the locations of the 1400 μL
stainless steel seat extension loop and seat capillary indicated………………….19
6. Chromatograms of EtG (0.08 μg/mL), EtG-D5, EtS(0.08 μg/mL) and EtS-D5 in a
spiked urine sample with a 900 μL injection volume with MRM
transitions ………………………………………………………………………..24
7. Chromatograms of EtG and EtS spiked into 1:1000 diluted blank urine and
separated with different ammonium acetate concentration in the 0.1% formic acid
mobile phase including: (A) 5 mM ammonium acetate, (B) 10 mM ammonium
acetate, (C) 15 mM ammonium acetate, and (D) 30 mM ammonium
acetate....…………………………………………………………………...….....27
8. MRM chromatograms of EtG, EtS, EtG-d5 and EtS-D5 in blank urine (A, C, E
and G) and spiked blank urine (diluted 1:1000) with a final concentration of 0.05
μg/mLfor each analyte (B, D, F and H) ……………….…………………….…..31
9. Representative calibration curves in deionized water and 1:1000 dilute blank
urine for EtG and EtS ……..........................................................…..………...….33
10. LC-ESI-MS/MS chromatograms in urine sample. (A) dilute reference certified
urine containing 0.050μg/mL EtG and EtS, and (B) blank urine that was not
spiked with either EtG or EtS……..……………………………………..………40
LIST OF TABLES
Table
Page
1. Retention Times (tR) and MS/MS parameters of EtG, EtS and internal standards,
declustering voltage, and collision energies used for acquisitions………………16
2. Average signal-to-noise (S/N) ratio of EtG and EtS in urine diluted 1:10 up to
1:1000 (n=3)………………….…………………………………………………..29
3. Average absolute peak area for EtG and EtS in deionized water and 1:1000
diluted urine………………………………………………………….…………..35
4. Accuracy and intra-day and inter-day precision for quantification of EtG and EtS
in 1:1000 diluted urine at three concentrations (0.050, 0.150, 0.500 μg/mL)
determined for calibration curve prepared in deionized
water……………………………………………………….…………………..…37
5. Accuracy and intra-day and inter-day precision for quantification of EtG and EtS
in 1:1000 diluted urine at three concentrations (0.050, 0.150, 0.500 μg/mL)
determined for calibration curve prepared in diluted blank
urine……………………………………………………………….………...…...38
1
1. Introduction
Alcohol and Alcohol Abuse
Alcohol is the most commonly-abused, addictive substance (1) and causes serious
public problems for people and countries. Alcohol abuse is a growing problem with
relevant health, clinical and social implications all over the world. For example,
driving under the influence of alcohol (DUI) is among the most serious of social
problems (2). Thus, alcoholism, as one of the most frequent addictions, is of great
interest in clinical and forensic medicine, and research efforts on ethanol consumption
biomarkers is a growing field in forensic toxicology (3).
Confirming alcohol (ethanol) intake is a major task in forensic toxicology and
psychiatry (4). Measurement of ethanol in serum, blood, hair or urine is the standard
way to detect alcohol consumption or monitoring abstinence (5). However, ethanol
itself is rapidly eliminated from the body (6). Measurement of ethanol is limited to
detecting only very recent consumption. Developing biomarkers of acute and chronic
ethanol consumption with longer half lives in the human body and lower detection
limits are needed.
Alcohol biomarkers
Testing of alcohol biomarkers is becoming an effective way to prove ethanol
consumption. Most of the ethanol consumed is oxidized to acetaldehyde and acetic
2
acid, but a small part undergoes non-oxidative transformation (Figure 1) (6). In
addition to carbohydrate deficient transferring, gamma glutamyl transferase, or mean
corpuscular volume, numerous researchers focus on non-oxidative ethanol
metabolites as biomarkers (7). Non-oxidative biomarkers include fatty acid ethyl
esters (FAEEs), β-D-ethyl glucuronide (EtG), phosphatidyl ethanol (PEth), ethyl
sulfate (EtS), and ethyl phosphate (EtP) (8-11). These biomarkers can be detected in
various biological samples for extended periods following ethanol consumption. For
example, FAEEs are detected in serum and blood up to 24 hrs after alcohol
consumption (8). EtG exists in urine for up to 5 days and PEth can be detected in
blood for more than two weeks following ethanol consumption (9). EtS and EtP, as
newly developed biomarkers, have been under investigation in recent years (12-14).
Unchanged in breath, urine and sweat
< 5%
< 0.1%
CH3CH2OH
Sulfotransferas
UDP-glucuronosyl
Ethyl glucuronide
(EtG)
-transferase (UGT)
Ethanol
(SULT)
> 95%
Ethyl sulfate
(EtS)
Alcohol and Aldehyde
dehydrogenase
CH3CHO + CH3COOH
Acetaldehyde and Acetic acid
Figure 1. Elimination pathways for ethanol in the human body, showing the major,
oxidate route, and the minor conjugation reactions with sulfate and glucuronic acid
3
Although the above mentioned alcohol biomarkers are detected in many body
fluids such as serum (15), sweat (16), blood (17) and urine (18) as well as hair (19),
the most widely used and effective way for studying alcohol exposure monitoring is
through urinary testing (20). Compared to ethanol, EtG and EtS are excreted in urine
for prolonged periods of time, making them useful as sensitive urinary alcohol
biomarkers.
EtG is the first direct metabolite of ethanol to be found in human hair in 1993 by
Sachs (21) and later by Schmitt et al (22). EtG (Figure 1 and 2) is a nonvolatile,
water-soluble, polar, and relatively stable molecule that can be detected in biological
fluids and tissues. It is formed by ethanol conjugation with glucuronic acid and is
mediated by UDP-glucuronyl tranderases, a superfamily of highly polymorphic
enzymes that are found mainly in the endoplasmic reticulum (23). It is estimated that
only 0.02 to 0.06% of the ingested ethanol dose is recovered as EtG in human urine
(24). Previous studies performed in human subjects indicated that the elimination of
EtG occurred over a longer period of time than that of the parent compound, ethanol.
Being a direct metabolite of alcohol, EtG is considered a specific marker of recent,
short-term alcohol consumption that can be detected in urine up to 80h after alcohol
consumption while the ethanol can no longer be detected (25). Therefore, the
development of a rapid and reliable procedure for measuring EtG in urine is of great
interest for forensic studies, as well as for traffic investigations involving alcohol
consumption.
4
Another minor direct ethanol metabolite, EtS (Figure 1 and 2), is a newly
discovered alcohol biomarker (26). EtS is formed by transfer of a sulfuric group from
3’-phosphoadenosine -5’-phosphosulfate to ethanol and is mediated by the family of
sulfotransferases (27). Animal studies indicated that EtS is excreted in urine mainly
during the first 24h, but can be excreted in urine for up to 30h (28). Because of the
difference in the metabolic formation of EtG and EtS, each can be used to indentify
recent ethanol consumption. Furthermore, the analysis of EtS, in addition to EtG, can
be used as further confirmation of ethanol consumption. In addition, recent research
suggested that bacterial urinary tract infection has less of an impact on EtS
determination because EtG is more susceptible to bio-degradation in the urinary tract
(29).
Figure 2. Chemical structures and molecular formulas of EtG and EtS and pentadeuterated internal standards
EtP, another newly discovered alcohol metabolite, has also been proposed to be
used as a urinary biomarker (30). However, EtP was measured at elevated
5
concentrations for only a very short period of time (31). Therefore, EtP is a less
sensitive biomarker than EtG or EtS and, to date, the use of EtP as a urinary
biomarker of alcohol consumption is not reported, because its half life is roughly
equivalence to that of ethanol.
Thereby, EtG and EtS, which have similar urinary excretion profiles, are
increasingly being used as urinary biomarkers indicating recent alcohol consumption.
In urine, EtG was detectable for up to 10 times longer than ethanol, while EtS was
detectable for up to 3~8 times longer than ethanol. Bicker et al. found a statistically
significant linear correlation between EtG and EtS concentration in urine (32), which
indicated they were both direct ethanol metabolites and had overlapping excretion
time profiles in urine. Simultaneous measurement of EtG and EtS in urine is
becoming an effective tool for monitoring alcohol consumption (20).
In addition to use as biomarkers for alcohol consumption, EtG and EtS can also be
regarded as high-specificity markers of alcohol exposure. Some recent reports about
alcohol exposure indicated that alcohol may be absorbed into the body from the use
of alcohol-containing mouthwash or frequent applications of alcohol-based hand
sanitizers, and that EtG or EtS may consequently be present in the urine of individuals
who only have these unintentional or accidental exposures to alcohol (33, 34).
Department of Health and Human Services has advised that specificity studies at low
EtG levels are needed for distinction of alcohol consumption and incidental alcohol
exposure. Many of the studies often conclude that the presence of EtG and EtS are
6
indicative of monitoring alcohol consumption or exposure during withdrawal
treatments or of situations where it is important to exclude alcohol use (eg.
Pregnancy), but fall short of developing an accurate method to analyze the extent of
alcohol intake in such a situation. The detection of small changes in concentrations of
alcohol metabolites is becoming a necessity for the effective interpretation of alcohol
exposure data. Therefore, it is important to develop a robust method for the
quantification of EtG and EtS at the low parts per billion levels in urine to establish
both the existence of alcohol consumption and the potential of other routes of alcohol
exposure.
Determination of EtG and EtS
The first analytical method for analyzing EtG as a biomarker of ethanol
consumption was reported by Schmitt et al.in the mid-1990s and was based on gas
chromatography
mass
spectrometry
(GC-MS)
(22).
The
method
required
derivatization to convert EtG, the highly polar glucuronide conjugate, to a substance
of higher volatility. The applicability of this technique to routine analysis remains
uncertain because of the lack of validation based on the published method.
Recently, non-GC-MS methods were developed and validated for the analyis of
EtG and EtS including liquid chromatography mass spectrometry or tandem mass
spectrometry (LC-MS or LC-MS/MS) (35), liquid chromatography with pulsed
electrochemical
detection
(36),
capillary
zone
electrophoresis
(37),
and
7
enzyme-linked immunosorbent assay (38). However, the non-MS methods are less
selective than the MS-based methods (39).
Comparing to other published methods, measurement of EtG and EtS by
LC-MS/MS is a rapid and sensitive method that does not require time-consuming
sample preparation procedures (such as derivatization) and is the technique of choice
for measuring EtG and EtS simultaneously. However, depending on the amount of
alcohol consumed, the time between drinking and urine sampling, and diuresis (24),
the EtG and EtS concentration may vary considerably between, and within,
individuals. It is necessary to remove interfering matrix constituents or, in case of
urinary analysis, to pre-concentrate the analytes prior to analysis by LC-MS/MS.
Current analytical published methods include sample pretreatment steps such as solid
phase extraction (SPE) (40-43), “evaporate and reconstitute” (44, 45) and “dilute and
inject” (32, 46, 47) approaches in connection with LC-MS/MS. SPE is commonly
reported in analytical methodology for urinary analyses (40). Despite the advantages
of SPE including commercial availability, concentration and clean-up of sample,
some disadvantages exist with SPE like variable recoveries of analytes, introduction
of potential interfering SPE extracts into the system, and the time and cost associated
with performing SPE. Though “evaporate and reconstitute” approach affords efficient
concentration, the complicated preparation and uncertain mass loss duing evaporation
limit its application. “Dilute and inject” approach is developing as an effective way of
cost effectiveness and rapid analysis for minimizing sample preparation steps in
8
recent years. Previous studies used a 1:2 to 1:50 dilution of urine with deionized
water, methanol, acetonitrile, or ammonium acetate buffer. However, matrix effects
still existed with dilution factors up to 50 (46) and a loss of sensitivity occurred due
to limited sample injection volume (32). In addition, for the determination of alcohol
metabolites with high polarity, such as EtG and EtS, it is very challenging to establish
a reliable LC-MS/MS method because these polar compounds show poor retention on
traditional columns prior to mass spectrometric detection. There are several reports on
the determination of low parts per million levels of EtG and EtS in biological fluids
and tissues in particular, for urine (42, 44-49). Although a wide variety of methods
were used, and the detection limits are not always provided, the concentrations of EtG
or EtS range from 0.100 μg/mL (44) to as high as 20 μg/mL (45). Recent reports for
EtG in urine range as 0.05 μg/mL (LOD) to 5.0 μg/mL (46). So far, this is the lowest
detection limit reported for EtG in urine available in the open literature.
Large-volume injection
Large-volume injection (LVI) has been used for the analysis of illicit and legal
drugs at low concentrations (50). Large volume injection (LVI) is a technique that
dates back to the early 1980s (51) and involves the direct injection of liquid samples
larger (30 – 2000 μL) than the conventionally-injected volumes of 10 - 20 μL. The
injection of a large volume of solvent of low elutropic strength containing the sample
effectively acts as the initial mobile phase, thereby concentrating analytes onto the
9
head of the analytical column. The injection solvent (e.g., water or non-aqueous
solvents), salts, and other matrix components do not partition into the stationary phase
and flow, un-retained, through the column to waste rather than to the detector (52).
After the injection phase, which is analogous to the sample concentration phase of
SPE, the elutropic strength of the mobile phase is increased to elute and separate the
concentrated analytes (53). The direct injection of aqueous sample volumes on an
analytical column is similar to frontal chromatography. With LVI, a large sample
volume (30-2000 μL), relative to the void volume of the column, is introduced
continuously rather than as a small volume band at the head of the column. As a
result, with LVI of aqueous samples, the low elutropic strength of the co-injected
water entering the LC column results in focusing of the analytes at the head of the
analytical column.
The benefits of LVI are recognized for a variety of applications including the
determination of phenylthiohydantoin-derivatized amino acids (54), biogenic amines
(55), trace analysis of pesticides, and herbicides and fungicides in vegetables and
soils (56). Additionally, LVI has been used for the determination of micropollutants
in surface waters, including pesticides (57), herbicides (58), fungicides (59), and
fluorinated alkyl substances (60). Previous work in our laboratory demonstrated the
use of large volume injection (e.g., 1800 μL) for the analysis of illicit and legal drugs
(61), polar organic contaminants such as fluorochemicals (62) in wastewater, and for
the quantification of fullerenes using normal-phase LC (63).
10
LVI offers several advantages including increased sensitivity and accuracy and
minimal sample handling since SPE is eliminated. In LVI, analyte pre-concentration
and analytical separation are linked automatically by adding one or more stainless
steel sampling loops to liquid chromatographs equipped with conventional,
commercial auto-samplers that are capable of injecting volumes in the hundreds of
microliters. Sample throughput is increased, at minimal cost and handling, compared
to SPE because no SPE cartridges or standalone hardware are needed. In addition, the
total sample volume required is smaller for LVI than for SPE because the entire
sample is injected, whereas only a fraction of off-line SPE extracts are typically
injected. Furthermore, method sensitivity can be improved when LVI is introduced
into “dilute and inject” approach. Thus, using LVI coupled with direct injection of
diluted urine instead of SPE as the sample cleanup procedure will be discussed and
applied to investigate an improved analytical method for the analysis of EtG and EtS
in urine.
Summary
The present work was aimed to develop and validate a rapid and sensitive
analytical method based on LC-ESI-MS/MS and large-volume injection (900 μL) for
the measurement of EtG and EtS in highly diluted urine. For this purpose, both
chromatographic and instrumental parameters were optimized. Different urine
dilution factors were tested and evaluated in order to minimize matrix effects. The
method was optimized and evaluated by determining accuracy and precision through
11
the testing of spiked urine samples. The detection and quantification limits of the
instrument and method detection limits were then determined using the optimized
conditions. Matrix effects were eliminated by using 1:1000 dilution of urine with
deionized water. Finally, the method was demonstrated and validated using
third-party reference urine with certified EtG and EtS concentrations.
2. Experimental
2.1 Standards and Reagents
Ethyl glucuronide (analytical grade >99%) and its deuterated standard, ethyl glucuronide-D5 (analytical grade >99%), were purchased from Cerilliant Corporation
(Round Rock, TX) at concentrations of 1 mg/mL in methanol. Ethyl sulfate sodium
salt (analytical grade >99%) and its deuterated standard, ethyl sulfate-D5 sodium salt
(analytical grade >99%), were obtained from AthenaES (Baltimore, MD).
HPLC grade methanol was purchased from Fisher Scientific (Fair Lawn, NJ).
Formic acid was purchased from EMD Chemicals (Gibbstown, NJ).
Ammonium
acetate was purchased from Mallinckrodt Chemicals (Phillipsburg, NJ). The mobile
phase of a buffer system of 0.1% formic acid and 15 mM ammonium acetate and 5%
methanol was prepared daily and filtered through hydrophilic polypropylene
membrane filters 0.45 μm purchased from Pall Corp (Ann Arbor, MI).
12
2.2 Preparation of Stock and Working Solutions
Individual standard stock solutions were prepared by dilution into methanol and
stored in the dark at -80◦C. The internal standards, EtG-D5 and EtS-D5, were
prepared by dissolution of each compound in methanol at the concentration of 100
μg/mL. EtG and EtS working stock solutions were further prepared in methanol at the
concentrations of 0.4, 1, 16, and 400 μg/mL by dilution. The working solutions were
stored in the dark at -20 ◦C and showed no appreciable degradation over a two month
period, with a maximum of ten freeze-and-thaw cycles. Small quantities of the
standard and internal standard working solutions were kept at 4 ◦C for daily analysis
and were replaced every two weeks.
Two sets of calibration curves were prepared and determined by adding appropriate
amounts of EtG and EtS standard solutions and 60 μL each of EtG-D5 and EtS-D5 (1
μg/mL) as internal standards. One calibration curve set was prepared in deionized
water and the second set of calibration standards were prepared in 1:1000 diluted
blank urine. Each calibration curve consisted of eight points for EtG (0.015, 0.05, 0.1,
0.25, 0.5, 1, 5, and 10 μg/mL,) and eight points for EtS (0.025, 0.05, 0.08, 0.1, 0.25,
0.5,1 and 5 μg/mL). The EtG and EtS concentrations correspond to 0.015-10 mg/mL
and 0.025-5 mg/mL in undiluted urine. The linearity of the calibration curve was
determined by plotting peak area ratios of the analytes to internal standards against
nominal concentrations of the analytes and was estimated by a 1/x weighing factor.
13
Instrumental blanks consisted of deionized water containing no standards or
internal standards. EtG and EtS standards at three concentration levels: 0.05
μg/mL(low), 0.15 μg/mL (medium), and 0.5 μg/mL(high) were used as quality
control samples. Blanks and quality control samples were run within each sequence to
monitor carryover, instrument background and sample preparation and all blanks
during the study were blank (no signal above baseline or at an S/N > 3).
2.3 Sample Preparation
Blank Urine Toxicology Control
A blank freeze-dried urine toxicology control sample was purchased from UTAK
under the name DAU (Lot # 1502) that was certified as free of EtG and EtS (UTAK
Laboratories, Valencia, CA, USA). It came as a dried material that was used as the
reference blank urine. The dried blank UTAK control material was reconstituted by
dissolving the material into 10 mL of deionized water as per the manufacturing
instruction. The urine control material was stored in the dark at -20 ◦C.
Spiked blank urine samples were prepared freshly following a standard procedure:
appropriate amount of the working standard solution of EtG and EtS was added to the
reconstituted blank urine sample. It was then diluted with deionized water based on
dilution factor of 1:1000. After dilution, a 2 mL aliquot volume of diluted blank urine
was then centrifuged in an Eppendorf centrifuge 5415 C for 30 min at a maximum
speed of 14,000 rpm (10,000 g). An aliquot of 1.2 mL of supernatant was then
14
transferred into a 2 mL autosampler glass vial and spiked with 60 μL of each
stable-isotope labeled internal standard to give a final concentration of 50 μg/L of
EtG-D5 and EtS-D5. The vials were transferred to an Agilent 1100 sample tray and
analyses were carried out within 24 hrs of sample preparation.
Certified UTAK Urine Control
The UTAK urine toxicology control with certified EtG and EtS concentrations of
50.0 mg/L (Lot# 3204), was purchased and received as a liquid and used as an
external reference standard for quality control purposes (UTAK Laboratories,
Valencia, CA, USA). The certified UTAK urine control sample was stored in the dark
at -20 ◦C. The certified UTAK urine control sample with a known EtG and EtS
concentration was diluted directly with deionized water based on dilution factor of
1:1000 and processed as described above.
2.4 Instrumentation
2.4.1 High Pressure Liquid chromatography (HPLC)
HPLC analyses were performed with an Agilent 1100 series system (Agilent
Technologies, Santa Clara, CA). The HPLC instrumentation was composed of a
vacuum degasser, a binary pump, and auto-sampler maintained at 4 ºC. Large-volume
injections and separations were conducted on this system that had been modified by
the addition of a commercially-available 900 μL “Injection Upgrade Kit” (Agilent
15
part no. G1363A) that consisted of a 900 μL analytical head, a 900 μL stainless steel
sample loop extension, and a 900 μL needle.
Chromatographic separation was carried out using an Agilent Zorbax-SBAq
column (150╳4.6 mm I.D., 5μm particle size) equipped with 2.0╳4.0 C18 security
guard column (Phenomenex,Torrance, CA) to protect the separation column. The
column was kept at 30 ºC during analysis. The flow rate was 500 μL/min. The
injection volume was 900 μL, and the mobile phase consisted of a buffer system of
0.1% formic acid and 15 mM ammonium acetate (A) and 5% methanol (B) in
isocratic mode for a total run time of 18 min.
2.4.2 Electrospray Tandem Mass spectrometry (ESI-MS/MS)
Detection and quantification were performed on a Waters Micromass tandem mass
spectrometer (Milford, MA) with an electrospray ionization (ESI) source operated in
negative ionization and multiple reaction monitoring (MRM) mode. Nitrogen and
argon were used as nebulizing and collision gases, respectively. The following
conditions were used in the mass spectrometer: source temperature, 150 °C;
desolvation gas temperature, 450 °C; capillary voltage, 3.57 kV; extractor voltage, 3
V; RF lens voltage, 0.2 V; cone gas flow, 25 L/h; desolvation gas flow, 650 L/h; ion
energy, 0.5 V; ion energy, 2.0 V; and entrance and exit potential, 11 V and 25 V. An
interchannel and scan delay of 0.03 s between groups of transitions was used to
enhance MS sensitivity.
16
Table 1. Retention times (tR) and MS/MS parameters of EtG, EtG-D5, EtS and
EtS-D5, declustering voltages (V), and collision energies (eV) used for acquisitions
Analyte
tR (min)
MRM pair
Declustering
Collison
Voltage (V)
Energy
(eV)
EtG
7.61
221.1/75 .0*
-20
−15
221.1/84.9
-20
-15
221.1/113.2
-20
-15
EtG-D5
7.61
226.0/75 .0*
-20
-15
EtS
8.87
125.0/96.9*
-20
-15
125.0/79.9
-20
-25
130.0/97.9*
-15
-15
130.0/79.9
-20
-30
EtS-D5
8.87
* MRM pairs for quantification
A total of eight transitions were acquired to quantify EtG and EtS analytes with
appropriate MRM parameters (Table 1). The mass transitions 221→75.0, 221→84.9
and 221→113.20 were selected for EtG while 226→75.0 was selected for EtG-D5
(internal standard). For EtS, 125.0→96.9 and 125.0→79.9 were selected for EtS
while 130→97.9 and 130.0→79.9 were selected for EtS-D5 (internal standard). The
17
transitions m/z 221→75.0 and m/z 226→75.0 were used to quantify EtG and EtG-D5,
respectively. The transitions m/z 125→96.9 and m/z 130→97.9 were used to quantify
EtS and EtS-D5, respectively. The remaining transitions were used as visual checks
for confirmation. The peak retention times of EtG and EtS at 7.61 min and 8.87 min,
given in the Table 1, were used to determine the appropriate time windows for each
group of transitions monitored in the MRM mode.
The characteristic precursor ions of EtG and EtG-D5 in ESI negative mode are the
deprontonated molecular ions [M-H]- m/z 221 and m/z 226, respectively, and are
consistent with ions observed by others (18,32). The most abundant product ions for
EtG (m/z 75.0, 84.9, 113.2) and EtG-D5 (m/z 75.0) may come from the fragmentation
of glucuronic aicd. The characteristic precursor ions of EtS is m/z 125 and the product
ions are m/z 96.9 [HSO4]- and m/z 79.9 [SO3]- . The precursor ion of EtS-D5 is m/z
130 and product ions are m/z 97.9 [DSO4]- and m/z 79.9 [SO3]- (Figure 3 and Figure
4).
18
(A) EtG
(B) EtG-D5
Figure 3. Product-ion spectra of EtG (A) and EtG-D5 (B)
(A) EtS
(B) EtS-D5
Figure 4. Product-ion spectra of EtS (A) and EtS-D5 (B)
19
2.5 Injection Volume and Dilution Factor Optimization
Five injection volumes (100, 500, 900, 1200 and 1400 μL) of EtG and EtS
standards in deionized water were tested to determine the optimum injection volume.
To reach a capacity of more than 900 μL, a commercially-available 1400 μL stainless
steel seat extension loop (Agilent part no. G13G13-87308) was installed between the
seat capillary fitting and port 5 (injection valve) of the analytical head (Figure 5).
Figure 5. Modified injection of an Agilent 1100 HPLC with the locations of the
1400 μL stainless steel seat extension loop and seat capillary indicated.
The 1,400 μL seat-extension loop was not necessary for sample injection volumes
of 900 μL or less. For injection volumes of 1,200 μL or greater, a injection progam
was initiated. A needle wash was followed by the withdrawal of 600 μL of sample
20
that was injected into the 1,400 μL seat capillary. This step was followed by the
withdrawal of an additional 600 μL to 800 μL to give a total sample injection volume
of 1,200 μL and 1,400 μL. For all analyses, the divert valve, located after the
analytical column and before the ESI interface, was switched to waste for the last five
min of each chromatographic analysis to protect the detector from unwanted material.
To minimize the matrix effects of urine, different dilution factors of urine were
tested. Blank urine samples, spiked with EtG and EtS, were diluted with deionized
water by five different dilution factors (1:10, 1:50, 1:200, 1:500, and 1:1000
(Vol/Vol)) in order to obtain a final concentration of 050 μg/mLfor EtG and EtS and
analyzed by LC-MS/MS using a 900 μL injection volume.
2.6 Specificity
Method specificity was checked by analyzing blank urine samples spiked either
with analytes in the absence of IS and with IS in the absence of analytes. Eight
different samples: four blank urine sample (neither analytes or IS), two blank urine
sample spiked with EtG-D5 and EtS-D5 only, and two blank urine sample spiked
with 0.05 μg/mL of EtG and EtS and no IS were analyzed by the optimized LVI of
900 μL and a dilution factor of 1:1000 to identify possible interfering peaks in the
urine matrix or standards.
2.7 Stability
After sample preparation, samples may remain in an auto-sampler tray for up to 24
hr before direct injection into the LC-ESI-MS/MS system for analysis. In order to
21
investigate potential degradation during this time period, spiked urine samples were
analyzed over a 24 hr period at three concentration levels (0.050, 0.150, 0.500 μg/mL,
n=3 each). The immediate analysis results (time = 0 h) and the EtG and EtS absolute
peak areas were used as the reference. The peak area of the analyte was plotted
against time to determine if the EtG or EtS degraded in the auto-sampler tray at room
temperature. The mean peak area at each time point was not significantly different
from the initial time point during the 24 hr period. These findings confirmed that both
EtG and EtS were stable during the 24 hr analysis period at room temperature and
were with regard to EtG and EtS complying with earlier data (35, 46).
2.8 Matrix Effects
The matrix effect of diluted urine (1:1000) was evaluated by analyzing 1:1000
diluted spiked blank urine with EtG and EtS, at four concentration levels including
0.01, 0.05, 0.25 and 1.0 μg/mL. Raw peak area of EtG and EtS were compared to raw
peak area for standards in deionized water at equivalent concentrations. The
concentrations of analytes in diluted urine corresponded to 10, 50, 250 and 1000
μg/mL in undiluted urine.
2.9 Validation
Accuracy, defined as recovery, was determined by spiking three replicates of
1:1000 diluted urine containing 0.05, 0.15, and 0.5 μg/mL each of EtG and EtS.
These three concentrations corresponded to concentrations in undiluted urine of 50,
150, 500 μg/mL.
22
Intra-day precision, calculated as the relative standard deviation (RSD), was
determined for three replicates (n=3) at each of three concentrations (0.050, 0.150,
0.5 μg/mL) prepared in 1:1000 diluted urine samples on a single day. Inter-day
precision was determined by analyzing three replicates of spiked urine aliquots at
same concentrations, prepared freshly on a daily basis over four consecutive days
during a single week. The concentrations of the analytes used to determine accuracy
and precision corresponded to low, medium and high concentrations used in the
calibration curves
Instrument detection limits (IDL) for EtG and EtS, defined as the concentration
needed to achieve a signal/noise (S/N) ≥ 3, were determined by analyzing standards
in deionized water ranging from 0.001 μg/mL to 0.05 μg/mL based on linear
regression. The method limit of quantification (LOQ), defined as the concentration in
diluted urine that gave a S/N ≥10, were determined by analyzing spiked 1:1000 dilute
urine samples that varied in concentrations from 0.005 μg/mL to 0.05 μg/mL.
3. Results and Discussion
3.1 Method Development
23
An effective sample cleanup (like SPE) and analyte enhancement from urinary
matrix was considered to be difficult and presumably lengthy because of the high
polarities of EtG and EtS, and the presence of inorganic and organic anions in urine
with similar extraction or chromatographic properties. Therefore, a high-throughput
analytical method based on direct injection of large sample volume of diluted urine
was developed. This strategy required improvements in chromatography in order to
sufficiently separate EtG and EtS from urinary matrix components. Thus, the effects
of the chromatographic column, mobile phase composition, injection volume and
sample dilution factors on chromatographic performance and overall method
performance were determined.
3.1.1 Chromatography
In this research, a new commercially available Agilent ZORBAX SB-Aq,
silica-based reversed phase LC column was used to increase the chromatographic
retention of EtG and EtS (Figure 6). EtG and EtS are polar compounds (Figure 1),
making them amenable to separation on an LC column with a hydrophilic stationary
phase. Satisfactory separation of EtG and EtS in diluted (1:1000) spiked blank urine
were achieved using a 900 uL injection volume on the Zorbax SBAQ column. The
instrument run times were 18 min, including LC column washing, separation and
re-equilibration. This column retained EtG and EtS to a greater extent than other
conventional reversed phase columns (33, 62). The ZORBAX SB-Aq column has a
hydrophilic surface that inhibits phase collapse, even in 100% aqueous mobile phases.
24
EtG 221.1-75.0*
EtG 221.1-84.9
EtG 221.1-113.2
EtG-D5 226.0-75.0*
%
EtS 125.0-96.9*
EtS 125.0-79.9
EtS-D5 130.0-97.9*
EtS-D5 130.0-79.9
2
4
6
8
10
Time (min)
12
14
16
Figure 6. Chromatograms of EtG (0.08 μg/mL), EtG-D5, EtS (0.08 μg/mL) and
EtS-D5 in a spiked urine sample with a 900 μL injection volume with MRM
transitions indicated.
Previously, published methods describe the use of reversed phase LC columns that
gave poor chromatographic retention or the use of self-made LC columns that are not
commercially available for routine analysis. For example, Helander and Beck (14)
used a Hypercarb column for which the retention of EtG and EtS was insufficient and
eluted close to the void volume. Dresen (64) used a polar-endcapped phenylpropyl
reversed phase column that gave good retention of the analytes with EtG eluting at
4.1 min and EtS eluting at 4.8 min. However, this method required a postcolumn
25
addition of acetonitrile to enhance the ESI sensitivity. Other previously published
methods for separating EtG and EtS include an acryamido-type hydrophilic column
(TSKgel Amide-80) with a split of the eluate (44), an octadecylsilyl column
(Chrompack Inertsil ODS-3) with isocratic elution (47), and a self-made mixed-mode
RP/weak anion exchange stationary phase based on N-(10-undecenoyl)-3amomioquinuclidine bonded to thiol- functionalized silica particles with isocratic
elution (32).
3.1.2 Optimization of Mobile phase
Initial mobile phase compostion consisted of aqueous 0.1% formic acid (vol/vol),
which gave retention times of 9.2 min for EtG and 11.3 min for EtS. Given the
selectivity of the mass spectrometer, this chromatographic separation was adequate.
However, variable retention times were observed when the mobile phase contained
only 0.1% formic acid. The addition of ammonium acetate was explored in order to
increase the pH of the buffer, and to improve peak shape while maintaining sufficient
chromatographic separation of EtG and EtS. Therefore, the effect of adding
ammonium acetate to the mobile phase was using 5 mM to 30 mM ammonium
acetate in the 0.1% formic acid buffer (Figure 7).
26
(A ) 5 mM
EtS
EtG
%
2
4
6
10
8
12
14
Time (min)
(B) )10mM
EtS
EtG
%
2
4
10
8
6
12
14
Time (min)
(C) 15 mM
EtS
%
EtG
2
4
6
8
Time (min)
10
12
14
27
(D) 30 mM
EtS
%
EtG
2
4
6
8
10
12
14
Time (min)
Figure 7. Chromatograms of EtG and EtS spiked into 1:1000 diluted blank urine
and separated with different ammonium acetate concentration in the 0.1% formic acid
mobile phase including: (A) 5 mM ammonium acetate, (B) 10 mM ammonium
acetate, (C) 15 mM ammonium acetate, and (D) 30 mM ammonium acetate.
Chromatographic retention times (tR) were reproducible with the addition of 10
mM or greater ammonium acetate. Higher ammonium acetate concentrations resulted
in a decrease in the mass spectrum base peak intensity, while lower ammonium
acetate concentrations resulted in decreased chromatographic resolution of EtG and
EtS. Therefore, as a compromise between sensitivity and selectivity, 15 mM
ammonium acetate was added to the aqueous 0.1% formic acid mobile phase.
Although tailing of the EtS peak (Figure 7) was observed, the retention of EtG
improved its detection by an almost one order of magnitude, as indicated by increased
signal-to-noise ratios (S/N), because of less background noise in the detection
28
window. With the addition of 15 mM ammonium acetate, the retention times of EtG
and EtS were 7.5 min and 8.6 min, respectively. This improved performance maybe
due to the increase pH or ionic strength. In addition, the ammonium acetate may mask
the silanol moieties on the stationary phase.
3.1.3 Optimization of Injection Volume
Using 0.1% formic acid with 10 mM ammonium acetate as the mobile phase and a
ZORBAX SB-Aq LC column, peak shape and resolution of EtG and EtS were
satisfactory and the injection volume could be optimized. Upon injecting five
different urine sample volumes including 100, 500, 900, 1200 and 1400 μL, it was
determined that peak shape of the analytes was symmetrical, with little or no evidence
of band broadening when the sample injection volume was 900 μL or less. In addition,
the EtG and EtS retention times were reproducible for 900 μL injection volumes.
However, when sample injection volumes were increased to 1200 μL or more, the EtS
peak shape became broad and tailed. Furthermore, there was a breakthrough of EtS
observed with injection volumes of 1400 μL. In order to obtain the best
reproducibility and sensitivity, 900 μL was chosen as the final injection volume and
was used for all subsequent experiments and analyses. A 900 μL injection volume is
up to 180 fold higher than that of the pervious studies that ranged from 5 μL to 15 μL
(32, 44, 46, and 64).
29
3.1.4 Optimization of Sample dilution factor
Urine’s high and variable ionic strength prevented direct injection without sample
dilution. A research goal was to optimize the dilution factor of the urine sample to
allow for injection of the greatest sample volume or concentration on the analytical
column possible without deteriorating chromatographic separation or increasing the
background noise (or decreasing the S/N).
Table 2. Average signal-to-noise (S/N) ratio of EtG and EtS in urine diluted 1:10 up
to 1:1000 (n=3).
S/N ratio
Dilution factor
EtG
EtS
10
3.8
ND*
50
10.3
8.5
200
63.2
54.1
500
166.7
99.6
1000
198.2
121.5
* ND represents not detectable
The EtG and EtS S/N ratios were compared at the same analyte concentration of
0.050 μg/mL by spiking EtG and EtS after the urine sample was diluted by five
30
different dilution factors from 1:10 to 1:1000 (vol/vol) (Table 1). In this experiment,
the final concentration and therefore the mass injected using a 900 μL injection
volume was constant while the matrix varied. The S/N values increased from 3.8 to
198.2 for EtG and from not detected to 121.5 for EtS when increased the dilution
factor from 10 to 1,000. The increase in S/N for higher dilutions while keeping the
mass injected constant indicated greater sensitivity. Therefore, a dilution factor of
1:1000 was chosen in order to maximizing sensitivity while minimizing matrix
effects. While a higher dilution factor was accompanied by a loss in the sensitivity,
Bicker et. al. (32) combined two different sample dilution factors 1:20 and 1:1000 as
a balanced approach to minimize the matrix effects as well as obtaining enough
sensitivity for effective measurement of real urine samples. However, from my study
sensitivity was well maintained because of the large injection volume of 900 μL used
with the high 1:1000 dilution factor and these conditions were used for all subsequent
experiments and analyses.
3.2 Method Validation
3.2.1 Specificity
The specificity or selectivity of the analytical method, the ability of an analytical
method to differentiate or quantify the analyte in the presence other components in
the sample, was determined (Figure 8). Chromatograms in Figure 8 show the MRM
31
chromatograms of blank urine, blank urine plus IS, and blank urine spiked with 0.050
μg/mL EtG and EtS. Chromatograms of A, C, E and G indicate the absences of EtG,
EtS, EtG-D5 and EtS-D5 in the blank urine while chromatograms of B, D, F and H
show the presences of each analyte. There was no significant interference caused by
endogenous substances from the urine in the EtG, EtS, EtG-D5 or EtS-D5 retention
time windows. These results indicate that this analytical method provided acceptable
specificity while the analytes were well distinguished and separated.
(A) 221→75
(C) 125→97
(E) 226→75
(G) 130→98
(B) 221→75 EtG
(D) 125→97 EtS
(F) 226→75 EtG-D5
(H) 130→98 EtS-D5
Figure 8. MRM chromatograms of EtG, EtS, EtG-d5 and EtS-D5 in blank urine (A, C,
E and G) and spiked blank urine (diluted 1:1000) with a final concentration of 0.05
μg/mLfor each analyte (B, D, F and H).
32
3.2.2 Linearity
Two calibration curves were constructed in (1) deionized water and (2) 1:1000
diluted blank urine. The ratio of the peak area of the analyte to that of the IS was
proportional to concentration of the analyte from 0.015 to 10 μg/mL for EtG, and
0.025 to 5 μg/mL for EtS. A linear regression model was used to describe the
regression relationship and different weighing factors (1/x, 1/x2, and none) were
examined for the best linear fit of the calibration curve. A 1/x weighing factor was
applied. Calibration curves with good linearity of the quantification of EtG and EtS in
deionized water and diluted blank urine samples were shown in Figure 9.
100
diluted blank urine
deionized water
80
area)
Response factor (Analyte area/IS
120
60
(A)
40
20
0
0
1
2
3
4
5
6
7
EtG concentration (μg/mL)
8
9
10
11
33
Response factor (Analyte area/IS
area)
100
90
80
diluted blank urine
70
deionized water
60
50
40
(B)
30
20
10
0
-0.5
0.5
1.5
2.5
3.5
4.5
5.5
EtS Concentration (μg/mL)
Figure 9. Representative calibration curves in deionized water and 1:1000 dilute
blank urine for EtG (A) and EtS (B).
Representative equations obtained with deionized water were y=11.224 x + 0.5977
(R2=0.9992) (EtG), y=17.396x + 0.1255 (R2=0.9998) (EtS) while typical equations
with diluted blank urine were y=11.131x + 0.5836 (R2= 0.9996) (EtG), y= 17.251x 0.1878 (R2= 0.9997) (EtS). Typical coefficients of determination (r2) for EtG and EtS
were ≥ 0.999. The linear range (0.015 to 10 μg/mL for EtG and 0.025 to 5 μg/mL for
EtS) were selected to represent the range of EtG (15 to 10,000 μg/mL) and EtS (25 to
5,000 μg/mL) concentrations observed in urine from subjects who have ingested
alcohol (17, 32, and 48).
34
3.2.3 Matrix effects
Endogenous compounds in the urine matrix can suppress or enhance ionization of
the analyte. Matrix effects may impair method reliability and limit precision, accuracy
and reproducibility. Ion suppression or enhancement is very common in atmospheric
pressure sources, especially ESI. Previous studies for the analaysis EtG and EtS in
urine using LC-ESI-MS/MS indicate matrix effects but that are normally
compensated for using internal standards (45, 46, 48, and 64).
Matrix effects were estimated by comparing the absolute chromatographic peak
area of each analyte spiked in deionized water to the equivalent concentrations in
spiked urine. In equation (3), Aurine is the absolute peak area of the analyte in the
spiked urine, and Awater is the absolute peak area of the analyte in deionized water.
Ion suppression or enhancement (%) = (Aurine - Awater) / Awater × 100
(3)
In Table 3, a positive value in the ion suppression/ enhancement column indicates that
diluted urine induced ion enhancement effects, while negative value indicates the
presence of ion suppression effects. By diluting the urine with a dilution factor of
1:1000, matrix effects, either ion suppression or enhancement, were overall
minimized to less than 8.33% (EtG) and 10.46% (EtS), which is comparable to the
inter-day precision in Table 4 and 5.
35
Table 3. Average absolute peak area for EtG and EtS in deionized water and 1:1000
diluted urine.
Analyte concentrations
Awater a
Aurine a
(μg/mL)
EtG
EtS
a
Statistically
Ion suppression or
equivalentb
enhancement (%)c
0.01
560±80
607±63
Yes
8.33
0.05
2124±190
2291±223
Yes
7.87
0.25
12601±890
12024±917
Yes
-4.58
1.0
52148±1147
52429±537
Yes
1.63
0.01
1524±111
1683±94
Yes
10.46
0.05
8576±166
8427±214
Yes
-1.73
0.25
41813±1997
38413±2501
Yes
-8.13
1.0
154580±2193 159094±2212
Yes
2.92
Mean ± 95% CI (n=3); b based on t-test ; C Calculations based on mean value of absolute peak area of
spiked blank urine samples versus the analyte response from deionized water (n=3);
Furthermore, the mean absolute peak areas of EtG and EtS in diluted urine were
statistically equivalent at the 95 % CI to those in deionized water, which indicated
that the large volume injection of a 1:1000 dilution eliminated matrix effects. Because
36
matrix effects were eliminated, quantification of EtG and EtG in diluted urine can be
performed using calibration standards prepared in deionized water. The eliminated
matrix effects for 1:000 diluting were also demonstrated by Bicker et. al. (32)
showing a deviation less than 5% for the mean absolute peak area of IS compared to
that of water. However, the calibration standards were still prepared in dilute blank
urine during their research because of the insufficient sensitivity with the small
injection volume, which brought the additional process and cost.
3.2.4 Accuracy and Precision
Accuracy and intra- and inter-day precision were determined by analyzing replicate
spiked diluted urine samples at three different EtG and EtS concentrations (0.05, 0.15,
and 0.5μg/mL).
For purposes of comparison, accuracy, as determined by EtG and
EtS recovery, was computed using equation (1) from calibration curves prepared in
deionized water and dilute blank urine. The precision, as indicated by relative
standard deviations (RSD, %), was calculated using equation (2) as the standard
deviation of the measured concentration relative to the measured concentration,
expressed as a percent of the standard deviation divided by the mean of measured
concentrations.
Accuracy (%) =
(Conc. measured- conc. spiked)/conc. spiked ×100
(1)
Precision (RSD, %) =
Standard deviation of conc. measured/conc. measured × 100
(2)
37
Table 4. Accuracy and intra-day and inter-day precision for quantification of EtG and
EtS in 1:1000 diluted urine at three concentrations (0.050, 0.150, 0.500 μg/mL)
determined for calibration curve prepared in deionized water.
Analyte
EtG
EtS
a
Nominal
Concentration
Accuracy b
Intra-day
Inter-day
conc.
measured.a
(%)
precisionb (%)
precisionb (%)
(μg/mL)
(μg/mL)
0.050
0.050±0.006
99.9
5.1
4.5
0.150
0.150±0.004
99.8
1.2
8.3
0.500
0.509±0.019
102
1.5
11
0.050
0.051±0.002
102
1.6
5.8
0.150
0.148±0.005
98.7
1.3
11
0.500
0.501±0.037
100
3.0
8.7
Mean ±95% CI (n=3);
b
n=3
The results summarized in Table 4 and Table 5 indicate good precision and
accuracy for the measurement of EtG and EtS in human urine based on two
calibration curves using this analytical method. Furthermore, concentrations of EtG
and EtS determined from these two calibration curves prepared in deionized water
and diluted blank urine were equivalent at the 95% CI. Therefore, the method was
accurate with overall recoveries ranged from 99.8% to 103% for EtG and 98.7% to
38
102% for EtS (Table 4). When quantifications from calibration curve prepared in
diluted blank urine were performed, similar accuracies were obtained (Table 5).
Quantification can be performed using calibration curves prepared in deionized water.
Table 5. Accuracy and intra-day and inter-day precision for quantification of EtG and
EtS in 1:1000 diluted urine at three concentrations (0.050, 0.150, 0.500 μg/mL)
determined for calibration curve prepared in diluted blank urine.
Analyte
EtG
EtS
a
Nominal
Concentration
Accuracy b
Intra-day
Inter-day
conc.
measured.a
(%)
precisionb (%)
precisionb (%)
(μg/mL)
(μg/mL)
0.050
0.050±0.006
101
5.0
4.6
0.150
0.154±0.014
103
3.7
8.3
0.500
0.499±0.024
99.8
1.9
14
0.050
0.047±0.012
93.3
10.2
6.4
0.150
0.147±0.007
98.2
2.0
1.6
0.500
0.512±0.031
103
2.4
3.2
Mean ±95% CI (n=3); b n=3
Intra-day precisions were less than 5.1% for EtG and less than 3.0% with an
average of 4.9% for EtS (Table 4). The inter-day precisions were less than 11% for
39
both EtG and EtS. Similar RSD values also obtained from the quantification based on
the calibration curve prepared in diluted blank urine (Table 5). The RSD values are
similar to those reported for the analysis of EtG and EtS in urine by LC-MS/MS
either using SPE-based technology at additional cost (40) or without using high
dilution and LVI at less sensitivity (32).
3.2.5 Detection and Quantification Limits
The instrument detection limit (IDL) was 0.005 μg/mL for EtG and 0.01 μg/mL for
EtS, which is a factor of 10 (or higher) lower than current published methodology for
EtG and EtS (14, 64).
The method LOQ was defined as the lowest concentration in the calibration curve
and that gave an S/N ≥10 in diluted urine. The LOQ was 0.015 μg/mL for EtG and
0.025 μg/mL for EtS in 1:1000 diluted urine, corresponding to 15 μg/mL for EtG and
25 μg/mL for EtS in undiluted urine. The LOQs determined for the LVI method
presented here are in the same or lower values as previous methods that used either
SPE or low dilution (up to 1:50) as pretreatments for urine samples (14, 32, 40, 45, 46,
48,and 64).
3.3 Method Verification
The analytical method was used to measure EtG and EtS in UTAK urine control
samples with certified concentrations of 50 μg/mL. The chromatograms in Figure 10
showed the chromatographic comparison of EtG and EtS in the UTAK urine control
sample and blank urine samples.
40
EtG
EtS
(A) Urine control
(B) Blank urine
Figure 10. LC-ESI-MS/MS chromatograms of (A) dilute reference certified urine
containing 0.05 μg/mL EtG and EtS, and (B) blank urine that was not spiked with
either EtG or EtS.
The measured concentrations of EtG and EtS in 1:1000 diluted urine samples at
0.050 μg/mL (corresponding to an initial concentration of 50 μg/mL) matched the
UTAK certified concentration at the 95%CI. The measured concentrations for the
certified reference urine were 0.049 ± 0.013 μg/mL for EtG and 0.050 ± 0.003 μg/mL
for EtS based on calibration curve prepared in deionized water. 0.050 ± 0.015 μg/mL
for EtG and 0.049 ± 0.002 μg/mL for EtS were obtained using calibration curves
prepared in 1:1000 diluted blank urine. Equivalent concentrations of EtG and EtS at
41
the 95% CI were obtained from calibration curves prepared in deionized water and
diluted urine, which demonstrate the method accuracy using calibration curves
prepared in deionized water for a cost effective and quantitative analyses.
4. Conclusions
An analytical method for the simultaneous detection, quantification and
confirmation of EtG and EtS in human urine samples using LC-ESI-MS/MS with LVI
was developed, fully validated, and demonstrated. The method consisted of 900 μL
injection volume, isocratic LC separation, tandem mass spectrometric detection, and
quantification of EtG and EtS. The direct injection of 1:1000 diluted urine samples
allowed for an increase in sample throughput while eliminating matrix effects. No
interfering peaks were found throughout the experiments, indicating good specificity
of the HPLC-ESI-MS/MS method for the simultaneous measurement of EtG and EtS.
Selectivity was ensured by monitoring MRM transitions of the precursor ions for both
EtG and EtS. The instrumental detection limit was 0.005 μg/mL for EtG and 0.010
μg/mL for EtS, a factor of 10 lower than current published methodology for EtG and
EtS. The LOQ was 0.015 μg/mL for EtG and 0.025 μg/mL for EtS in 1:1000 diluted
urine, which corresponded to 15 μg/mL for EtG and 25 μg/mL for EtS in undiluted
urine. Finally, the method accuracy and precision were demonstrated by obtaining
statistically-equivalent concentrations to that for certified third-party reference urine
42
from a commercial source. The method was accurate, rapid, cost-effective and
reproducible without any sample pretreatment procedures.
5. Acknowledgements
The author thanks Edward Kim, Dawn Fulton and Kelly Lenore from Agilent for
HPLC column selections and method improvement in this work.
43
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