Article
Synephrine as a Specific Marker for Orange Consumption Matthias Bader, Tatjana Lang, Roman Lang, and Thomas Hofmann J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 22 May 2017 Downloaded from http://pubs.acs.org on May 25, 2017
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Journal of Agricultural and Food Chemistry
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Synephrine as a Specific Marker for Orange Consumption
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Matthias Bader†,‡, Tatjana Lang†,‡, Roman Lang†,‡, and Thomas Hofmann†§
4 5 6
‡
contributed equally
7 8
†
9
München, Lise-Meitner-Straße 34, D-85354 Freising, Germany,
Chair for Food Chemistry and Molecular Sensory Science, Technische Universität
10
§
11
85354 Freising, Germany.
Bavarian Center for Biomolecular Mass Spectrometry, Gregor-Mendel-Straße 4,
12 13 14 15 16 17 18 19
*
20
PHONE
+49-8161/71-2902
21
FAX
+49-8161/71-2949
22
E-MAIL
[email protected] To whom correspondence should be addressed
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ABSTRACT
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To validate the suitability of synephrine, known as a highly abundant alkaloid in
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oranges, as a dietary biomarker for orange consumption, a highly sensitive and
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robust stable isotope dilution analysis (SIDA) as well as an ECHO method, using the
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analyte itself as a pseudo-internal standard injected into the analysis run providing an
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“echo peak” of the analyte, was developed to quantitate synephrine by LC-MS/MS in
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citrus juices and human urine before and after ingestion of orange juice. A citrus juice
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screening revealed high synephrine concentrations of 150 to 420 nmol/mL in orange
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(n=11) and tangerine juices (n=2), while 20 to 100 times lower levels were found in
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juice from grapefruit (n=14), lemon (n=5) and lime (n=4), respectively. Application of
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the SIDA to quantitate synephrine in sulfatase/glucuronidase-treated urine samples
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(n=10) after orange juice consumption showed an increase of synephrine from trace
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levels (0.1 ±0.1 nmol/mL) in the 2-days washout phase to a maximum concentration
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of 8.9 (±5.5) nmol/mL found 4 h after ingestion of orange juice. While proline betaine
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has been recently reported as a dietary biomarker indicating the ingestion of any
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citrus product and Chinese artichoke, respectively, synephrine may be used a
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reliable additional biomarker with high specificity for orange and tangerine.
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Keywords: Dietary biomarker, synephrine, ECHO quantitation, citrus, oranges
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INTRODUCTION
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The beneficial impact of dietary patterns on human health is today investigated in
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epidemiological and human intervention studies, mostly by using questionnaires to
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assess types of food, quantities and regularity of intake. Although clear instructions
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for the study subjects as well as physician involvement have been shown to improve
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compliance to some extent, the subjective nature of self-reported dietary intake
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assessment methods presents numerous challenges to obtaining accurate dietary
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intake and nutritional status. Analytical monitoring of dietary biomarkers is considered
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a suitable strategy to overcome this limitation as it holds promise to objectively
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assess dietary consumption without the bias of self-reported dietary intake errors.
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The analytical compliance control of study subjects is, however, still limited by
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the lack of suitable biomarker molecules reflecting wider aspects of diet. For
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example, N-methylpyridinium ions have been identified as a urinary dietary biomarker
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indicating the intake of roasted coffee1 and the analysis of alkylresorcinols in plasma
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was reported to be suitable for measuring the consumption of whole grains.2,3 Very
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recently, quantitative high-throughput LC-MS/MS screening of a variety of foods and
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beverages confirmed extraordinarily high levels of proline betaine (1; figure 1), also
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known as stachydrin, in citrus fruits as well as in Chinese artichoke,4 thus confirming
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earlier reports.5-7 While the intake of other foods did not significantly affect the urinary
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excretion profiles, consumption of Chinese artichoke resulted in similarly high
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abundance of 1 in urine as found for citrus juice.4 As elevated urinary levels of 1 were
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found to be also indicative for the consumption of Chinese artichoke, proline betaine
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cannot be considered a citrus-specific dietary biomarker.
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In comparison, the alkaloid synephrine (2; figure 1), which has been found to
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act on α-, β1-, β2- and β3-adrenoceptors,8 has been reported as a trace amine present 3 ACS Paragon Plus Environment
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in bitter oranges,8-10 sweet oranges,11-13 tangerines,12-14 and lemons,12,13 while other
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foods seem not to contain significant amounts.15 This alkaloid has just recently been
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reported in orange honey and has been proposed as an authenticity marker of
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orange honey.16 While the (R)-configured synephrine is the naturally occurring
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enantiomer in fruits, traces of the (S)-isomer have been detected in thermally
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processed juices and marmalades.14 The isomeric phenylephrine, coined m-
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synephrine (3), seems not to occur naturally in citrus fruits and was only found in a
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few dietary supplements.17-21
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The objective of the present study was to develop a precise, sensitive and
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accurate LC-MS/MS method using an internal standard and an ECHO standard,22-24
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respectively, to enable a fast quantitation of 2 and 3 in various citrus fruits and dietary
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supplements, and to investigate its suitability as a candidate citrus-specific urinary
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biomarker.
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MATERIALS AND METHODS
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Chemicals. Synephrine
(2),
phenylephrine
(3,
as
hydrochloride salt),
d3-
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phenylephrine (d3-3), ammonium acetate, β-glucuronidase (from Helix pomatia, Type
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HP-2, ≥ 100000 U/mL), sulfatase (from Helix pomatia, Type H-1, ≥ 10000 U/g), d4-
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methanol and methanol (LC-MS grade) was purchased from Sigma-Aldrich
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(Steinheim, Germany). Water for LC-MS separation was purified with an integral 5
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system (Millipore, Schwalbach, Germany). Citrus samples and juices were obtained
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from a local supermarket (Freising, Germany). Citrus fruit (~2 kg) were individually
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squeezed, combined and homogenized. Fresh juices were frozen at -20°C until
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analysis.
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Urine Collection After Food Ingestion. Urine samples (frozen at -80°C) were
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taken from a human intervention study conducted in 2016.4 In brief, 27 healthy
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volunteers (age 24-32 years), with no reports on food-specific allergic reactions and
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drug intake, were asked to abstain from citrus products for at least two days (wash-
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out phase) and, then, to collect a morning urine sample. Ten of the volunteers were
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then asked to consume orange juice (250 mL), which was shown to contain 68 µmol
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(11.5 mg) synephrine (2). At least three further urine samples were collected on that
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day (day 0), then morning urine on the next day (day 1) and a sample the afternoon,
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finally a morning urine sample each on day two and three. For each specimen, the
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time-point of sampling was recorded to calculate the time passed after the ingestion
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of the food to enable plotting of synephrine/creatinine (pmol/µmol) versus time after
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ingestion (h). Individual data on urinary creatinine levels were taken from a recent
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report.4 The participants were allowed their own individual eating habits but were
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asked to abstain from any citrus product until after the final urine sample was
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collected.
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Quantitation of Synephrine (2) and Phenylephrine (3). Standard Solutions:
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synephrine (2; 5 mg) and phenylephrine (3; 5 mg as hydrochloride salt) were
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individually dissolved in d4-methanol (2 mL each) and aliquots (600 µL) used to
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accurately determine the concentration by means of quantitative
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spectroscopy.25 Aliquots of these stock solutions were combined to yield an analyte
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stock with 100 nmol/mL of each analyte.
1
H NMR
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Matrix Calibrations, Precision and Bias: The analyte stock was serially diluted
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with water to yield working solution containing 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 5 ACS Paragon Plus Environment
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0.78, and 0.39 nmol/mL of synephrine and phenylephrine, respectively. All of these
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dilutions (100 µL) were further spiked into matrix (blank urine or blank lemon juice,
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900 µL each) to yield matrix standard with final analyte concentrations 10000, 5000,
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2500, 1250, 1250, 625, 312.5, 156.3, 78.1, and 39.1 nmol/L in matrix. Aliquots of
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these matrix calibrations standards (100 µL) were mixed with the internal standard
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working solution of d3-phenylephrine (6000 nmol/L, 100 µL) and an aqueous
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ammonium acetate solution (10 mmol/L, 800 µL). The standards were injected in
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replicates (n=3) and calibration curves constructed from area ratios and
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concentration ratios. Precision and accuracy values of the matrix standards (cf. table
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1) are given from the back-calculated standards and calibration curve.
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Quality control samples were prepared (triplicates) similarly in urine (2500 and
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156 nmol/L) and lemon juice (2500 and 313 nmol/L). Precision was calculated as
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relative standard deviation of the mean concentration of the replicates. Bias was
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calculated as the ratio of the calculated and the nominal value.
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Enzymatic Hydrolysis of Urine Samples: The enzyme suspension consisted of
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sulfatase (100 mg) and glucuronidase (suspension, 0.1 mL) in water (1 mL). To
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liberate synephrine from phase I/II conjugates, urine samples (100 µL) were mixed
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with the enzyme suspension (50 µL) and incubated for 4 h at 37 °C. After hydrolysis,
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the internal standard solution (6.0 mmol/L, 50 µL) was added. After vortexing (5 s),
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acetonitrile/methanol (9/1, v/v; 800 µL) was added, the suspension mixed and
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centrifuged (10 min, 4 °C, 13200 rpm). The supernatant was decanted into new
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Eppendorf caps and dried under a stream of nitrogen at 37 °C. The samples were
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dissolved in aqueous ammonium acetate (10 mmol/L, 50 µL) and analyzed by means
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of LC-MS/MS.
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Statistics. Data handling was done using Microsoft Excel 2016 and GraphPad
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Prism versions 5.00 and 7.00 for Windows (GraphPad Software, La Jolla California 6 ACS Paragon Plus Environment
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USA, www.graphpad.comGraphpad Prism). Synephrine/creatinine ratio (pmol/µmol)
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in spot urine samples were determined for the segments 0-2, 2-4, 4-8, 8-12, 12-24
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and 24-36 h to calculate means and standard deviations. Synephrine/creatinine
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values of morning urine prior to the food intervention study (blank) were subtracted
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from the morning urines collected after orange juice consumption, followed by
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Wilcoxon
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synephrine/creatinine value in the morning urines taken before and after food
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consumption.
signed
rank
test
to
evaluate
significant
differences
between
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Instrumental Analysis with HPLC-MS/MS. The chromatographic system was
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a Shimadzu Nexera X2 UPLC (Shimadzu, Duisburg, Germany), comprising an
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Autosampler (SIL 30AC, kept at 15 °C), pumps (2×LC30AD), degasser (DGU 20
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A5R), column oven (CTO 30A, kept at 40°C) and communication device (CBM 20A).
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The UPLC was connected to an AB Sciex 5500 Qtrap mass spectrometer (Sciex,
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Darmstadt, Germany) operating in positive electrospray mode. Analyst 1.6.2 was
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used for instrument control. Curtain Gas was 40, collision gas “medium”, ion spray
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voltage -5.5 kV, source temperature 500 °C, nebulizer gas 50 and heater gas 60.
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Resolution was set to “unit”. Dwell time for each mass transition was 20 ms. At least
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two mass transitions per compound were recorded. The samples were separated on
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a Kinetex Phenyl-F5 column (1.7 µm, 100×2.1 mm, Phenomenex, Aschaffenburg,
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Germany) with ammonium acetate in water (10 mmol/L; eluent A) and ammonium
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acetate in methanol (10 mmol/L; eluent B) at a flow rate of 300 µL/min. After
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injection, eluent B was increased from 3 – 80% in 3 min with a non-linear gradient
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(curve 8) followed by 1 min of isocratic elution. The starting conditions were re-
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established within 0.5 min and equilibration was 2.5 min prior to the next injection.
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The column effluent was diverted to the MS source between1 – 5 min. 1 min after
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sample injection, the ECHO standard (100 pmol/mL, 2 µL) was injected. 7 ACS Paragon Plus Environment
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RESULTS AND DISCUSSION
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Method
Development
for
Quantitation
of
Synephrine
(2)
and
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Phenylephrine (3). The method development involved the preparation of standard
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solutions of the analytes synephrine (2) and phenylephrine (3) in deuterium oxide
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and exact quantification of the concentration by quantitative 1H NMR to obtain stock
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solutions which were diluted appropriately. Detection of the analytes was facilitated
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by tuning the ion source and ion path parameters of the MS/MS system in positive
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electrospray for optimized collision induced dissociation. One quantifier and two
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qualifier mass transitions were recorded for each analyte (table 1).
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The chromatographic separation of the stable isotope dilution analysis (SIDA)
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using HPLC-MS/MS was performed on a phenyl-F5 column using d3-phenylephrine
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(d3-3) as the internal standard for quantitation of analytes 2 and 3. In comparison,
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quantitation was performed by means of the ECHO technique using purified
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synephrine (2) as the pseudo-internal ECHO standard.23,24 A time interval of 1 min
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after sample injection, a defined amount of the purified analyte (2) was put onto the
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column by means of a second injection to navigate the ECHO peak, which served as
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a pseudo-internal standard and was used to normalize the analyte signal, in close
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proximity to the analyte, that is ~1.3 and ~0.4 min after synephrine (2) and
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phenylephrine (3), respectively (figure 2). The total run-time was 6.5 min including
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equilibration time and retention times were stable in both citrus juice and urine matrix
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(cf. table 1). The method therefore was fast enough to handle the samples from the
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orange juice drinking study in a reasonable time. 8 ACS Paragon Plus Environment
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Calibration curves were calculated and recovery experiments were performed
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in analyte-free lemon-juice and urine to evaluate and compare the quantitation
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methods using the SIDA and the ECHO method, respectively. The analysis of
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analyte-free lemon juice spiked with 2 and 3 (figure 2, A) and authentic orange juice
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(B) showed well shaped and resolved peaks of the analytes, the internal standard
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and the ECHO standard, respectively. The lower end of the calibrated range of 2 was
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78 nM in urine and 156 nM in juice matrix, and 156 nM and 78 nM in urine and juice
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for 3, respectively (table 1), providing sufficient sensitivity for analysis of trace
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amounts in urine and citrus juice within our studies. The replicate analysis of lemon
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juice spiked with two different concentrations of the analytes 2 and 3 gave precision
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values
