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A General LC-MS/MS Method for Monitoring Potential β-Lactam

Contamination in Drugs and Drug-Manufacturing Surfaces

Article  in  The AAPS Journal · July 2018

DOI: 10.1208/s12248-018-0224-7

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The AAPS Journal (2018) 20:70
DOI: 10.1208/s12248-018-0224-7

Research Article

A General LC-MS/MS Method for Monitoring Potential β-Lactam

Contamination in Drugs and Drug-Manufacturing Surfaces

Chen Qiu,1 Hongbin Zhu,1,2 Connie Ruzicka,1 David Keire,1 and Hongping Ye1,3

Received 5 March 2018; accepted 3 April 2018

Abstract. Penicillins and some non-penicillin β-lactams may cause potentially life-
threatening allergic reactions. Thus, possible cross contamination of β-lactams in food or
drugs can put people at risk. Therefore, when there is a reasonable possibility that a non-
penicillin product could be contaminated by penicillin, the drug products are tested for
penicillin contamination. Here, a sensitive and rapid method for simultaneous determination
of multiple β-lactam antibiotics using high performance liquid chromatography-tandem mass
spectrometry (LC-MS/MS) was developed and validated. Mass spectral acquisition was
performed on a Q-Exactive HF mass spectrometer in positive ion mode with parallel reaction
monitoring (PRM). The method was validated for seven β-lactam antibiotics including one or
two from each class and a synthetic intermediate. The quantification precision and accuracy
at 200 ppb were in the range of ± 1.84 to ± 4.56 and − 5.20 to 3.44%, respectively. The limit of
detection (LOD) was 0.2 ppb, and the limit of quantitation (LOQ) was 2 ppb with a linear
dynamic range (LDR) of 2–2000 ppb for all eight β-lactams. From various drug products, the
recoveries of eight β-lactams at 200 and 2 ppb ranged from 93.8 ± 3.2 to 112.1 ± 4.2% and
89.7 ± 4.6 to 110.6 ± 1.9%, respectively. The application of the method for detecting cross
contamination of trace β-lactams (0.2 ppb) and for monitoring facility surface cleaning was
also investigated. This sensitive and fast method was fit-for-purpose for detecting and
quantifying trace amount of β-lactam contamination, monitoring cross contamination in
manufacturing processes, and determining potency for regulatory purposes and for quality
control.
KEY WORDS: cross contamination; LC-MS/MS; parallel reaction monitoring; penicillin; β-lactams.

INTRODUCTION or run-off from agricultural use and landfills. Cases of allergic

reactions after consumption of foods containing antibiotic
Penicillin or other non-penicillin drugs containing a β- residues have been widely reported in the literature (8–11).
lactam moiety are antibiotics with a long history in the treatment Thus, antibiotic monitoring is an essential quality control
of a wide range of infectious diseases in both human and measure in safe food or drug production.
veterinary applications. While these antibiotics play an impor- The massive production of antibiotics has created
tant role in disease treatments, they pose a potential risk for another risk—cross contamination. Cross contamination of
individuals who are hypersensitive to them (1–4). In fact, β-lactams, especially exposure of non-penicillin products to
penicillin allergy is the most common cause of drug-induced penicillins, has been shown to be a safety concern for patients
anaphylaxis, and the allergy accounts for up to 1000 deaths per (6,12–14). Recent cases involving potential cross contamina-
year (5–7). Hundreds of thousands of tons of antibiotics are tion in repackaging operations, loss of containment in
consumed each year, and 30–60% pass through animals and segregated facilities, potential undeclared penicillin in ho-
humans completely unchanged. The different compounds then meopathic products, and contaminated compounded products
reach the ocean via hospital waste, municipal sewage, fish farms, have required the use of test methods stipulated by regula-
tions. However, the current FDA’s guidance for industry (i.e.,
1 BNon-Penicillin Beta-Lactam Drugs: A CGMP Framework
Division of Pharmaceutical Analysis, Center for Drug Evaluation
for Preventing Cross-Contamination^) (15) references a test
and Research, U.S. Food and Drug Administration, 645 S.
Newstead Ave, St. Louis, Missouri 63110, USA. method which was published in 1965 and that lacks sufficient
2
Present Address: ThermoFisher Scientific Inc., 3747 N Meridian Rd, sensitivity to low levels of β-lactams (16). Because the
Rockford, Illinois 61101, USA. threshold dose at which allergenic response could occur is
3
To whom correspondence should be addressed. (e–mail: extremely low and difficult to define (11,17), a more sensitive
Hongping.ye@fda.hhs.gov) and robust analytical method is a critical need in the food and

1550-7416/18/0000-0001/0 # 2018 American Association of Pharmaceutical Scientists

70 Page 2 of 9 The AAPS Journal (2018) 20:70

pharmaceutical industries for regulatory purposes or for Optima LC-MS grade solvents were purchased from Fisher
product quality control. Scientific (Pittsburgh, PA). Milli-Q water (Millipore, MA)
Liquid chromatography (LC) with UV detection has been was used for preparation of all solutions.
used to determine antibiotics (18–20), but such methods often lack
the sensitivity and specificity required for β-lactam detection. Preparation of Samples, Calibration Standards, and
Thus, these LC-UV techniques have been replaced by methods Validation Samples
that use mass spectrometry (MS) detection to provide more
specific determination leading to unequivocal confirmation of the Stock solutions of each β-lactam standard were prepared
compounds studied. However, most of the LC-MS methods have at 400 ppm in the mobile phase mixture without formic acid
been developed for the analysis of residue penicillin and related (A/B, 10:90, v/v, see chromatographic conditions). The
antibiotics in food products (21–25). In addition, these methods solutions were sonicated for 5–30 min to ensure the solid
are not optimized or validated for trace amount detection in drug particulates fully dissolved. A 5% acetonitrile solution was
formulations. For example, Bekoe et al. used an LC-MS/MS used as the diluent for the preparation of the calibration
method to investigate the quality of antibiotic drug products with mixtures from the individual standard stocks. A stock mixture
potency measurements on 13 antibiotic active pharmaceutical containing 20,000 ppb of each β-lactam standard and 200 ppb
ingredients (26). However, the Bekoe et al. method was developed dPNG as an internal standard was prepared. Prior to LC-MS/
for the assay of antibiotics not for trace level determination. MS analysis, a series of dilutions were made from the
While working with an FDA compliance for-cause 20,000 ppb standard mixture to obtain concentrations of
assignment in 2015, a sensitive LC-MS/MS method for the 2000, 200, 20, 2, and 0.2 ppb for each standard. The final
detection and quantification of penicillin G (PNG) and 6- concentration of the internal standard dPNG was 200 ppb in
aminopenicillanic acid (6-APA) in homeopathic products was all standard solutions. These standard mixtures were used to
developed in our laboratory. In the current study, more obtain calibration curves and to determine the method’s limit
extensive method development and validation were con- of detection (LOD) and limit of quantitation (LOQ) for β-
ducted to extend the LC-MS/MS method for the determina- lactams. Two separate mixtures at 200 and 400 ppb were
tion of all five types of β-lactam antibiotics (27) found in prepared for all standards and used for instrument precision
different formulations, including tablets, capsules, suspen- and accuracy tests.
sions, and injections. Using this validated method, cross The recovery test samples were prepared by spiking
contamination and facility surface cleaning were also investi- eight β-lactam standards and the internal standard dPNG into
gated for selected drugs. This LC-MS/MS approach is a single solutions made from the drug products. For tablets and
method that can be used for simultaneous detection of capsules, composites were generated using the contents of ten
multiple β-lactams at levels as low as 0.2 ppb. Moreover, capsules or the powder from ten ground tablets. Samples
since a wide LC gradient was used and the drugs tested here having an equivalent amount of the active compound (i.e.,
were dispersed throughout the chromatogram, the method 10 mg according to the label claim) were transferred to a
should be easily adapted to other β-lactams and antibiotics. 10-mL volumetric flask and dissolved in 5 mL of 5%
acetonitrile. Samples were sonicated for 10 min and diluted
MATERIALS AND METHODS to volume with 5% acetonitrile. An aliquot of the test sample
was filtered using a 0.20-μm filter. For injection and suspen-
Materials, Reagents, and Standards sion formulations, the drug containers were shaken vigor-
ously for a minute, an amount equivalent to 10 mg of the
The β-lactam standards of amoxicillin (AMX), ampicillin active compound by volume was transferred to a 10-mL
(AMP), penicillin G (PNG), cefuroxime (CFX), imipenem volumetric flask and mixed with 5 mL 5% acetonitrile.
(IMP), loracarbef (LRC), and aztreonam (AZT) were Samples were sonicated for 10 min and diluted to 10 mL
purchased from USP (Rockville, MD). The 6- with 5% acetonitrile. An aliquot of the test sample was
aminopenicillanic acid (APA) was obtained from Santa Cruz filtered using a 0.20-μm filter. Filtrates were diluted 1000
Biotechnology, Inc. (Dallas, TX). The d7-penicillin G (dPNG) times while adding desired amounts of the standards. Three
and d4-amoxicillin (dAMX) were purchased from Toronto samples were prepared for each drug filtrate: a blank
Research Chemicals, Inc. (Toronto, Canada). All standards containing the internal standard only and two samples with
had purities greater than 99%. Drug products were purchased 2 and 200 ppb β-lactam standards, respectively, in addition to
through a pharmacy (Table I). Optima formic acid and the internal standard.

Table I. Drug Products Used in this Study

Names Formulations Dosages Manufacturer

Penicillin G potassium Injection 5,000,000 units SANDOZ

Amoxicillin Capsules 500 mg SANDOZ
Ampicillin Suspension for oral 125 mg/5 mL DAVA
Cefuroxime Injection 750 mg SAGENT
Penicillin V potassium Tablets 500 mg AUROBINDO
Lovastatin Tablets 40 mg LUPIN
The AAPS Journal (2018) 20:70 70 Page 3 of 9

Table II. Name, Structure, Molecular Weight (Mw), Water Solubility (Sw), PRM Transition, and Collision Energy for Standards

HCD
Mw Sw* Precursor ion
Name Chemical structure Energy
(g/mol) (g/L) quantifier
(eV)

(+)-6-
Aminopenicillanic acid 216.26 4.0 217.0640 189.0692 35
(APA)

Imipenem (IMP)
317.36 10.0 300.1009 126.0374 30

Amoxicillin (AMX) 419.45 3.93 366.1115 114.0012 30

Aztreonam (AZT)
435.43 0.043 436.0589 313.0599 30

Loracarbef (LRC)
367.78 0.325 350.0898 174.0550 30

Ampicillin (AMP)
349.40 0.605 350.0898 106.0656 30

Penicillin G potassium
(PNG) 372.48 100 335.1060 176.0706 35

Cefuroxime sodium
Freely
(CEF) 446.37 442.1024 211.0173 30
soluble

Penicillin G-d7
379.52 342.1496 183.1146 35
(internal standard)

Amoxicillin-d4
369.43 370.0373 212.0682 25
(internal standard)

*Solubility references: 1. ChemIDplus (US National Library of Medicine). 2. DrugBank database

70 Page 4 of 9 The AAPS Journal (2018) 20:70

Fig. 1. Chromatogram of standard solution containing eight β-lactams at 200 ppb with MS detection

For the cross contamination test, samples were prepared in samples were prepared by depositing 10 and 100 μL of
highly concentrated drug solutions. A 5% acetonitrile solution solutions containing 2 and 20 ng of PNG separately on
was added to the powder from tablets/capsules to form a slurry two thoroughly cleaned bench areas. After air drying, the
containing 100 mg/mL of the active compounds. The slurries bench surfaces were wiped five to eight times with small
were sonicated for 5 min followed by centrifugation at 3000 g for pieces of filter papers (~ 2 × 2 cm) wetted with 5%
30 min. The supernatants and injections (100 mg/mL active acetonitrile. The filter papers were then soaked in
compounds) were filtered using 0.20-μm filters. Two samples 10 mL of 5% acetonitrile solution to extract out the
were prepared for each drug filtrate: a blank containing the PNG. A control sample was prepared on a clean bench
internal standard only and a sample with 0.2 ppb β-lactam area where no PNG was deposited. All three solutions
standard mixture and an internal standard of 200 ppb. were filtered with 0.20-μm filters, and the internal
To mimic possible contamination in a manufacturing standard dPNG of 200 ppb was added to each testing
facility, a surface contamination test was designed. These sample prior to LC-MS/MS analysis.

Fig. 2. Chromatograms of all standards at 0.2 ppb extracted from PRM acquisition of a standard mixture,
with the count number labeled by each peak. AA refers to autointegrated area
The AAPS Journal (2018) 20:70 70 Page 5 of 9

Table III. Validation Parameters Summarized for Eight Investigated β-Lactams Determined Using Calibration Curves Within Linear Dynamic
Range (LDR) of 2–2000 ppb

APA IMP AMX AZT LRC AMP PNG CEF

2
r (2–2000 ppb LDR) 0.9878 0.9997 1 0.9999 0.9999 0.9998 0.9997 1
Accuracy (%) 200 ppb − 5.12 − 5.20 1.04 0.05 2.03 3.44 3.07 − 0.89
400 ppb 0.87 −1.50 5.84 5.54 0.92 − 0.80 3.25 0.67
Precision (std%) 200 ppb 1.84 3.34 4.56 2.69 3.86 2.19 2.71 3.72
400 ppb 5.34 4.48 3.01 4.21 2.86 2.63 3.24 4.39

Chromatographic and Mass Spectrometric Analyses system. A Waters Acquity UPLC BEH C18 (1.7 μm, 2.1 × 150
mm) column at 35°C was used for sample separation. Mobile
LC-MS/MS analysis was performed on a Thermo Q- phase A was water-acetonitrile-methanol at 90:6:4 (v/v/v), and
Exactive HF mass spectrometer coupled with a Vanquish LC mobile phase B was water-acetonitrile-methanol at 5:65:30 (v/v/

Fig. 3. Recoveries of eight β-lactams at 2 ppb from five different drug matrixes. The recoveries and standard errors were calculated based on
three injections
70 Page 6 of 9 The AAPS Journal (2018) 20:70

v). Both mobile phases contained 0.1% formic acid (98%). The and samples were normally Bdirty,^ to protect the instrument
LC gradient consisted of several steps starting with 1% (B) MS data was only acquired within short time windows when
holding for 5 min (0–5 min) and increasing to 75% (B) during the targeted compounds (e.g., PNG) eluted.
15 min (5–20 min). Isocratic elution at 75% (B) for 1 min (20– For each standard, collision-induced dissociation prod-
21 min) was followed by mobile phase A increasing back to 99% ucts of precursors were detected in the positive ion parallel
during 1 min (21–22 min) and equilibrating for another 8 min reaction monitoring (PRM) mode. To set up the PRM
(22–30 min). Flow rate was 250 μL min−1, and the injection experiment, selected precursor ion for each standard was
volume was 10 μL with the autosampler set at 4°C. added to Binclusion list^ with corresponding detection time
The mass spectrometer was operated in the positive ion window. PRM transitions and collision energies for each
mode and calibrated using an LTQ Positive Ion Calibration compound are listed in Table II. Peak area integration was
Mix (Pierce). Xcalibur™ instrument control software (version performed using the Thermo QualBrowser software.
4.0) was used for data acquisition. The key parameters were
set as follows: ionization voltage at 3.5 kV, sheath gas at 4 and RESULTS AND DISCUSSION
auxiliary gas at 1 arbitrary unit, and capillary temperature at
325°C. Mass scan range was 100 to 500 m/z with resolution of Method Development and Optimization
60,000 for MS1 and 30,000 for MS2. Data acquisition time was
23 and 18 min for MS1 and MS2, respectively. Since all For this work, the challenge in sample preparation was
compounds eluted before 16 min, the MS2 acquisition was the solubility differences of the eight β-lactam compounds. To
stopped at 18 min. LC elute was diverted to waste when not make stock solutions at the highest concentration possible and
at data acquisition. For cross contamination test samples, the sample solvent closest to mobile phase A possible, various
because the concentrations of the drug APIs were very high mixtures of water, methanol, and acetonitrile were investigated.

Fig. 4. Detection of trace amounts of β-lactams in drug products. a 0.2 ppb penicillin G
spiked into amoxicillin capsules: blue trace represents amoxicillin drug only, red trace
represents 0.2 ppb penicillin G spiked into amoxicillin drug. b 0.2 ppb amoxicillin spiked
into penicillin G injection: blue trace represents penicillin G only, red trace represents
0.2 ppb amoxicillin spiked into penicillin G
The AAPS Journal (2018) 20:70 70 Page 7 of 9

Fig. 5. Detection of penicillin G from a contaminated facility surface. Blue line represents the control area,
red line represents contamination of 0.2 ppb, and green line represents contamination of 2 ppb penicillin G

The mixture of mobile phases A and B at 10 to 90 was found to IMP. However, because APA and IMP have distinct
give clear solutions for all compounds at 400 ppm. Sonication precursor/quantifier ions (Table II), accurate quantification
was needed for compounds with low solubility. can be achieved despite non-baseline separation. IMP was
Although chemical structures, acid-base properties, and split into two peaks that have the same mass and fragments.
solubility of these targeted β-lactams are different, they are These peaks represent the presence of the imine and
all polar and reverse phase liquid chromatography was secondary amine in equilibrium. Thus, two peak areas were
suitable for separation on column. The best possible resolu- summarized for quantification of IMP.
tion was desired, because the LC-MS/MS method was to
simultaneously detect and quantify all five classes of β- Method Validation
lactams. Technically, these β-lactams can be analyzed with
both positive and negative ionization modes, or by switching Through properly designing the PRM experiment by
between two modes for different lactams. Here, to simplify assigning each precursor in a specific time window in the MS2
the mobile phase composition and to make method transfer inclusion list, the high specificity of detection for each β-
to a different LC-MS system more feasible, only the positive lactam was achieved. Comparing with selected ion monitoring
mode was chosen in the method development. (SIM), the low noise of the selective PRM method improved
Eight β-lactam standards were injected separately for the sensitivity of the method. Figure 2 shows chromatograms
LC-MS to optimize the chromatographic and mass spectro- of all eight β-lactams at a concentration of 0.2 ppb. All peaks
metric conditions and to obtain individual MS spectra. Then, had similar full width at half height and peak shape with
a mixture of eight standards was injected together for further sufficient ion counts for identification and quantitation. The
optimization and for appropriate selection of the precursor/ lowest concentration investigated in this study was 0.2 ppb
quantifier for each β-lactam standard (Table II). A typical which was defined as limit of detection (LOD).
chromatogram of the mixture is shown in Fig. 1. Figure 1 The peak area ratios of analytes to the internal standard
shows that most of the standards were well separated and were used for quantification to minimize the variations caused
dispersed throughout the chromatogram except APA and by instrument conditions, possible degradation or day to day

Table IV. Potency of β-lactams in Drug Products

Drug products Concentrations determined by assay test (ppb)

Amoxicillin (capsules) 1111.3 ± 33.4

Ampicillin (suspension) 899.0 ± 23.2
Penicillin G (injection) 1145.4 ± 31.5
Cefuroxime (injection) 1093.1 ± 25.8
70 Page 8 of 9 The AAPS Journal (2018) 20:70

differences. Calibration curves for all eight β-lactams were undoubtedly detected and identified. Figure 4a shows the
obtained from standard concentrations between 2 to 2000 ppb detection of 0.2 ppb PNG in amoxicillin capsules, and
and coefficients of determination (r2) were greater than 0.99 Fig. 4b shows the detection of 0.2 ppb AMX in a
for all β-lactams in linear fits of the data across this range. All penicillin G injection. Because the method was validated
results were the average of three injections. The lowest with a LOQ of 2 ppb, quantification to 0.2 ppb spiking
concentration of calibration curves, 2 ppb, was defined as the was not attempted. If desired and the observed peaks
limit of quantitation (LOQ). Instrument precision was have sufficient signal to noise, the amount of β-lactam
investigated using data from six injections of β-lactam present can be estimated using calibration curves. These
standard mixtures at 200 and 400 ppb. Precision was results demonstrate that the method was capable of
expressed as a percentage of standard deviation at two detecting and quantifying trace levels of β-lactams in
concentrations and was between 1.84 and 5.34 (Table III). various therapeutic formulations. Thus, the method can be
Method accuracy at 200 and 400 ppb for each β-lactam was used for both limit and quantification tests. To detect
also determined with six injections using calibration curves. trace levels of contaminates, the sample concentration of
These results indicate the method was suitable for simulta- the API must be very high, to the point where the API
neous quantification of all β-lactams from different classes. does not need to be completely dissolved. After sonication
The effect of drug excipients (matrix effect) was exam- of the highly concentrated sample, the trace amount of
ined by spiking β-lactam standards into different drug contaminates present should all be in solution. Filtering of
formulations including tablets, capsules, suspensions, and the saturated sample removed any remaining API partic-
injections (both intravenous and muscular). The standard ulates from the sample before analysis. Clear injectable
mixture at concentrations of 2 and 200 ppb each along with drugs can be analyzed Bas is^ after spiking with the
the internal standard dPNG was added to five drug matrix internal standard and filtering. For quantification purpose,
preparations, and the recovered amount was determined the deuterated internal standard solution should be made
using calibration curves. For each drug preparation, a blank freshly to reduce possible H/D exchange. Minor un-
sample spiked with dPNG only (no other β-lactam) was deuterated compound may appear if the stock internal
analyzed with LC-MS/MS and the background concentration standard solution is not fresh.
was subtracted from the spiked pool concentration to obtain
the recovery. For example, to determine spiked AMX
recovery in an amoxicillin injection, the amount of amoxicillin Detection of Facility Surface Contamination
(active ingredient) was determined from the blank sample.
This amount was subtracted from the spiked pool concentra- Therapeutics are generally manufactured in large production
tion for AMX, and the resulting concentration was compared plants and production lines for a range of different active
with a nominal concentration to obtain AMX recovery. At pharmaceutical ingredients are often run in parallel or share the
the concentration of 200 ppb, the recoveries were between same equipment. The facility should be tested for cleanliness when
93.8 ± 3.2 and 112.1 ± 4.2% for all eight β-lactams in all five switching to a different production line to prevent cross contam-
drug matrixes (data not shown). Figure 3 shows the recover- ination and to ensure the safety of the finished products. Two
ies of each β-lactam standard from five drug matrixes at 2 ppb cleaning test samples along with a blank sample were prepared
spiking, ranging from 89.7 ± 4.6 to 110.6 ± 1.9%. These results and analyzed with the β-lactam validated method. As shown in
demonstrated that the existence of drug excipients did not Fig. 5, 0.2 ppb of penicillin G was clearly detected from the
affect the quantification of β-lactams using this method. contaminated surface. For the 2 ppb contamination sample, the
experimental concentration of penicillin G was 2.24 ± 0.12 ppb
from three LC-MS/MS injections. The small peak in the control
Application of the Method
sample is the minor un-deuterated impurity in deuterated
penicillin G which was added as an internal standard at 200 ppb.
Detection of Trace Amounts of β-Lactams in Drug Products

The validated method was applied to detect trace Assay of β-Lactam APIs in Drug Products
amounts of β-lactams in drug products and cross contam-
ination between different β-lactam drugs. Two β-lactam Although this method was developed for the determination
drugs, amoxicillin capsules and a cefuroxime injection, at of trace levels of β-lactams, the method can also be used to
100 mg/mL API (as label stated) were spiked with 0.2 ppb measure the potency of β-lactam drug products since it was
PNG and 200 ppb dPNG as an internal standard. A validated for quantification in the range of 2–2000 ppb. As stated
symmetric sample, penicillin G injection at 100 mg/mL in the recovery study section, for each of the five drug
API spiked with 0.2 ppb AMX and 200 ppb dAMX as an preparations, a blank sample spiked with an internal standard
internal standard, was also analyzed to examine the only (no other β-lactam) was analyzed using LC-MS/MS and the
method’s flexibility. In addition, a non β-lactam drug, background concentration was subtracted from the spiked pool
Lovastatin tablets at 100 mg/mL API, was tested by concentration to obtain the recovery. The background concen-
spiking with 0.2 ppb PNG and 200 ppb dPNG to trations were API concentrations in the drug products. Those
demonstrate the capability of the method to detect trace drug preparations were made at 1000 ppb according to label
penicillin contamination in a non β-lactam drug product. claims. The concentration of penicillin G was estimated because
For all these four samples, a blank drug preparation the label claim was 5,000,000 units per vial and therefore had no
spiked with dPNG or dAMX only was analyzed for weight claim. Table IV shows the experimentally determined
comparison. In all cases, spiked β-lactams at 0.2 ppb were concentrations of the β-lactam API using the corresponding
The AAPS Journal (2018) 20:70 70 Page 9 of 9

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