Solid Phase Extraction Application notebook

dissolved in the mobile phase Recoverybefore HPLC analysis. RESULTS 6 HPLC Method with UV detection Column: Hypersil Gold C18 column 150mm x 2.1mm, 3µ...

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Solid Phase Extraction Application notebook

For Milk analyses

Q4 2013

INTRODUCTION POLYINTELL offers a comprehensive range of sorbents for the challenging fields of sample preparation, sample clean-up, extraction and purification, from conventional SPE products (AttractSPETM polymeric sorbents, SilactSPE silica based-sorbents) for a crude clean-up to the most innovative SPE for specific application (AFFINIMIP®SPE, a Molecularly Imprinted Polymers (MIP) based sorbents). In addition, the experience of POLYINTELL is continuously enriched with customer interactions and an endless analytical development for new applications. This experience is sometimes communicated through Application notes (available on website and via newsletters). To make you enjoy of this background and for an wealthy, very instructive and easily-accessed overview of AFFINIMIP®SPE and AttractSPETM performances, we are pleased to introduce you this Application Notebook which collects a good abstract of POLYINTELL’s experience on AFFINIMIP®SPE and AttractSPETM. For your convenience, this application notebook will be permanently updated with new protocols and results. Please regularly visit our website www.polyintell.com for the latest version of the Application Notebook. This Application notebook will be an essential tool to address your technical issues. TECHNICAL SUPPORT POLYINTELL has fully integrated technologies platform with specialized teams in organic chemistry, polymer chemistry, analytical and bioanalytical chemistry who are at your disposal to guide you in your challenges. At POLYINTELL, we are committed to providing the best technical support possible. Our Technical Support Group is a team of highly qualified M.Sc. and PhD Chemists, who are at your disposal to resolve your problem and to answer to your queries. For technical inquiries, feel free to contact us either by email: [email protected] , or by phone at: +33 (0)2 32 09 32 70. We are also very thankful to customer’s feedback about our products, protocols and customer services by email to: [email protected]

QUALITY POLICY To develop a long term and durable partnership with its customers, POLYINTELL ensures the best quality of its products and services. As an ISO9001:2008 certified company, POLYINTELL has implemented Quality management system requirements to show its commitment to quality, customers, and a willingness to work towards improving efficiency. In addition, to ensure the best quality of its products, the performance is checked by following several QC tests according to each product’s quality control procedure. After passing all these tests, the products receive a certificate of analysis which proved the compliance with the defined criterion.

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APPLICATIONS available for MILK analyses Milk is a very complex matrix which requires well suited sample preparation to monitor the presence of mycotoxins or chemical contaminants in order to prevent adulteration or health risks for the consumer. This specific application notebook contains a wide range of applications for MILK matrices. ANALYTES

AFFINIMIP® SPE ANALYTES

MATRICES

Bisphenol A

AFFINIMIP® SPE Bisphenol A

Milk (liquid and powdered infant formula)

REF

PAGE

FS106

4-5

Water Canned food Red and white wines Beer Human urine

Tetracyclines (CTC, OTC, TC)

AFFINIMIP® SPE Tetracyclines

Milk

FS112

6

Aflatoxin M1

AFFINIMIP® SPE Aflatoxin M1

Milk

FS116

COMING SOON!

Chloramphenicol

AFFINIMIP® SPE Chloramphenicol

Milk

FS110

7

Honey Bovine Urine Shrimp

Sulfonamides

AttractSPETM SCX

Milk

SCX25.S.3.60

7

Melamine

AttractSPETM SCX

Milk

SCX25.S.6.150

8

Cyanuric acid

AttractSPETM SAX

Milk

SAX25.S.6.150

8

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AFFINIMIP® SPE Bisphenol A DETERMINATION OF BISPHENOL A IN LIQUID INFANT FORMULA

RESULTS

Regulations for Bisphenol A: Europe (directive 2011/8/EU) : forbiden in infant feeding bottles

1100

1000

Injection 50µL of Infant Formula before treatment

900

800

700

600

mVolts

Bisphenol A after treatment of 15mL of Infant Formula

500

400

300

200

PROTOCOL OF PURIFICATION Sample preparation

100

0

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

Minutes

Chromatograms of Infant Formula containing 1µg/L of Bisphenol A before clean-up (Red) and after clean-up (Blue) with AFFINIMIP® SPE Bisphenol A.

Purification with a 3mL/100mg AFFINIMIP® SPE Bisphenol A cartridge

900

Equilibration •5mL Methanol -2% Acetic Acid •5mL Acetonitrile •5mL Water Loading Up to 15mL of infant formula Washing of interferents •10mL Water •6mL Water/Acetonitrile (60/40) Drying 30 seconds Elution (E) 3mL Methanol The elution fraction was then evaporated and dissolved in the mobile phase before HPLC analysis.

800

Bisphenol A

700

600

mVolts

500

400

300

200

100

0 9,0

9,2

9,4

9,6

9,8

10,0

10,2

10,4

10,6

10,8

11,0

11,2

11,4

11,6

11,8

12,0

12,2

12,4

12,6

12,8

Chromatograms obtained after clean-up with AFFINIMIP® SPE Bisphenol A of 15mL of Infant Formula spiked with Bisphenol A at 2µg/L (tested twice, blue) or at 1µg/L (tested twice, red) or not spiked (pink). Recovery of Bisphenol A in 15mL of infant formula after AFFINIMIP® SPE Bisphenol A clean-up and relative standard deviation calculated from results generated: - under repeatability conditions (n=3, % RSDr)

HPLC Method with Fluorescence detection Column: Hypersil Gold C18 column 150mm x 4.6mm Mobile phase: gradient profile Time (min) 0 2

% water 65 65

% ACN 35 35

12

50

50

20 20.5 35

50 65 65

50 35 35

Flow rate: 1mL/min Fluorescence detection: wavelengths: 230 / 315nm Injection volume: 50µL.

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13,0

Minutes

C° (µg/L)

Mean (µg/L)

Recoveries %

% RSDr

1.0

0.9

88.4

1.5

2.0

1.7

85.7

2.7

- under reproducibility conditions ( % RSDR). C° (µg/L)

Mean (µg/L)

Recoveries %

% RSDR

1.0

0.8

84.4

7.4

2.0

1.7

85.8

5.3

Catalog number: 3mL-100mg sorbent in a PP cartridge FS106-02 for 25 cartridges FS106-03 for 50 cartridges 6mL-100mg sorbent in a glass cartridge FS106-02G for 25 cartridges FS106-03G for 50 cartridges Link to AFFINIMIP® SPE Bisphenol A

excitation/emission

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AFFINIMIP® SPE Bisphenol A DETERMINATION OF BISPHENOL A IN POWDERED INFANT FORMULA RESULTS

Regulations for Bisphenol A: Europe (directive 2011/8/EU) : forbiden in infant feeding bottles

1400

Fluo 2-MD1.dat

Fluo 2-md2.dat

Fluo 4-md1.dat

Fluo 4-md2.dat

Fluo mnd.dat

1300

1200

1100

Bisphenol A

1000

900

800

PROTOCOL OF PURIFICATION Sample preparation 4.4g powdered infant milk was reconstituted in 30 mL of water and warmed up at ~ 50°C during 20 seconds using microwaves. Then 20 mL of acetonitrile were added to 20 mL of warm milk and centrifuged at 4000 rpm during 10 minutes. The supernatant was collected and filtered on filter paper (4-7µm). This extract was diluted 1:1 with water to form the loading solution. Purification with a 3mL/100mg AFFINIMIP® SPE Bisphenol A cartridge Equilibration •5mL Methanol -2% Acetic Acid •5mL Acetonitrile •5mL Water Loading Up to 40mL of infant formula Washing of interferents •10mL Water •6mL Water/Acetonitrile (60/40) Drying 30 seconds Elution (E) 3mL Methanol The elution fraction was then evaporated and dissolved in the mobile phase before HPLC analysis.

mVolts

700

600

500

400

300

200

100

0

-100 6,5

7,0

7,5

8,0

8,5

9,0

9,5

10,0

10,5

11,0

11,5

12,0

12,5

13,0

% water 65 65

% ACN 35 35

12

50

50

20 20.5 35

50 65 65

50 35 35

Flow rate: 1mL/min Fluorescence detection: wavelengths: 230 / 315nm Injection volume: 50µL.

www.polyintell.com

14,0

Chromatograms obtained after clean-up with AFFINIMIP® SPE Bisphenol A of equivalent at 10mL of Infant Formula spiked with Bisphenol A at 4.3µg/L (tested twice, red) or at 2.1µg/L (tested twice, blue) or not spiked (pink).

Recovery of Bisphenol A spiked at different concentrations after 3mL/100mg AFFINIMIP® SPE Bisphenol A clean-up of 40mL of loading solution (equivalent to 10mL of reconstituted Infant milk) and relative standard deviation calculated from results generated under repeatability conditions Concentration of BPA in reconstituted milk (µg/L)

Mean concentration (µg/L)

Recoveries %

RSDr %

2.1

2.3 (n=5)

108

8.7

4.3

4.0 (n=4)

95

3.7

HPLC Method with Fluorescence detection Column: Hypersil Gold C18 column 150mm x 4.6mm Mobile phase: gradient profile Time (min) 0 2

13,5

Minutes

Catalog number: 3mL-100mg sorbent in a PP cartridge FS106-02 for 25 cartridges FS106-03 for 50 cartridges 6mL-100mg sorbent in a PP cartridge FS106-02B for 25 cartridges FS106-03B for 50 cartridges 6mL-100mg sorbent in a glass cartridge FS106-02G for 25 cartridges FS106-03G for 50 cartridges Link to AFFINIMIP® SPE Bisphenol A

excitation/emission

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AFFINIMIP® SPE Tetracyclines DETERMINATION OF TETRACYCLINES IN MILK RESULTS

Regulations for Tetracyclines: Codex Alimentarius: 100µg/L sheep or cattle milk (26th CAC session, 2003)

60

OTC TC

50

CTC

40

mAU

PROTOCOL OF PURIFICATION Sample preparation 1.5mL of Milk were mixed with 6mL of EDTA/Mc Ilvaine’s Buffer and the mixture was centrifuged at 4000rpm for 10 minutes at a temperature below 15°C. The supernatant was collected and 750µL of a 1N NaOH solution was added and the solution was then adjusted to pH 10 with a NaOH solution (this mixture was the loading solution).

30

10 0

0.0

% 10mM Oxalic Acid ACN

% MeOH

0

90

5

5

20

90

5

5

21

80

10

10

40

80

10

10

41

90

5

5

15.0

20.0

25.0

30.0

35.0

40.0

45.0

50.0

Recovery of Tetracyclines spiked at 50µg/L after AFFINIMIP® SPE Tetracyclines clean-up of 1.5mL of Milk and relative standard deviation calculated from results generated under repetability conditions. Tetracyclines

C° (µg/L)

Mean (µg/L)

Recoveries % RSDr % (n=3)

Tetracycline

50.8

48.9

96.3

5.1

Oxytetracycline

50.7

47.3

93.3

2.7

Chlortetracycline

51.0

34.8

70

13.8

Recovery of Tetracyclines spiked at 50µg/L after AFFINIMIP® SPE Tetracyclines clean-up of 1.5mL of Milk and relative standard deviation calculated from results generated under reproducibility conditions. Tetracyclines

C° (µg/L)

Mean (µg/L)

Recoverie % RSDR s% (n=3)

Tetracycline

50

44.3

88.7

9.5

Oxytetracycline

50

53.7

107.3

10.7

Chlortetracyclin e

50

40.7

81.3

9.6

Catalog number: 1mL-10mg sorbent FS112-02A for 25 cartridges 1mL FS112-03A for 50 cartridges 1mL kit of 12 reservoirs 15ml and adapters for use with 1, 3 & 6 mL columns ACC-AR2 Link to AFFINIMIP® SPE Tetracyclines

Flow rate: 0.2mL/min UV detection: 355nm Injection volume: 100µL.

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10.0

Minutes

HPLC Method with UV detection Column: Hypersil Gold C18 column 150mm x 2.1mm, 3µm Mobile phase: gradient profile % 10mM Oxalic Acid Water

5.0

UV Chromatograms (355nm) obtained after cleanup with AFFINIMIP® SPE Tetracyclines of 1.5mL of Milk spiked with Tetracyclines at 50µg/L (blue) or not spiked (red) or of 1.5mL of water spiked with Tetracyclines at 50µg/L (pink)

Purification with a 1mL/10mg AFFINIMIP® SPE Tetracyclines cartridge Equilibration •1mL Acetonitrile •1mL Water Loading Load the loading solution (7.5mL) Washing of interferents •2mL Water/Acetonitrile (60/40) Drying 3 minutes Elution (E) 2mL Methanol with 2% Formic acid The elution fraction was then evaporated and dissolved in the mobile phase before HPLC analysis.

Time (min)

20

6

AFFINIMIP® SPE Chloramphenicol DETERMINATION OF CHLORAMPHENICOL IN MILK PROTOCOL OF PURIFICATION Sample preparation Whole milk is centrifuge during 15min at 5000 rpm. Loading solution is the intermediate layer or is the skimmed milk. Purification with a 1mL/50mg AFFINIMIP® SPE Chloramphenicol cartridge Equilibration •2mL Acetonitrile •2mL Water Loading 1mL of loading solution for 15µg/kg (or 10mL for 0.3µg/Kg) Washing of interferents (W1) •1mL Water •1mL (Water - 0.5% AA)/ACN (95/5) •2mL of Ammonia (1%) in Water •2mL (Water-1% Ammonia)/ACN (80/20) Drying 1 min Washing of interferents (W2) 0.25mL Diethyl ether Elution (E) 2mL Methanol The elution fraction was then evaporated and dissolved in the mobile phase before HPLC analysis.

Regulations for Chloramphenicol: Europe (2003/181/EC): prohibited with a minimum required performance limits of 0.3µg/Kg

Catalog number: 1mL-50mg sorbent FS110-02A for 25 cartridges 1mL FS110-03A for 50 cartridges 1mL Link to AFFINIMIP® SPE Chloramphenicol

AttractSPETM SCX DETERMINATION OF SULFONAMIDES IN MILK PROTOCOL OF PURIFICATION Purification with a 3mL/60mg AttractSPETM SCX cartridge Equilibration •2mL Methanol •2mL Water Loading 5mL Milk Washing of interferents •2mL Methanol/Water (5/95) •1mL 0.5M HCl •2mL Methanol/Water (20/80) Elution (E) 2.5mL Ammonium bicarbonate/Methanol (10/90) The elution fraction was then evaporated and dissolved in the mobile phase before HPLC analysis. Analyse LC/MS-MS

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Regulations for Sulfonamides: Sulfadimethoxine 0.01ppm in milk (U.S. FDA 21 CFR 556.640) Sulfaethoxypyridazine 0 in milk (U.S. FDA 21 CFR 556.650)

Catalog number: 6mL-150mg sorbent SCX-25.S.3.60 for 25 cartridges SCX-50.S.3.60 for 50 cartridges Link to AttractSPETM SCX

AttractSPETM SCX

DETERMINATION OF MELAMINE IN MILK PROTOCOL OF PURIFICATION Sample preparation Add 4mL water to 5g liquid infant formula or 1g dry infant formula. Shake during 10-20min with 20mL 50/50 ACN/Water and centrifuge for 10minutes at 3400 rpm. The supernatant is the loading solution. Purification with a 6mL/150mg AttractSPETM SCX cartridge Equilibration •5mL 0.1M NaOH in Acetonitrile •5mL 0.1M HCl in Acetonitrile •5mL Acetonitrile •5mL 4% Formic acid in Water Loading 3mL 4% Formic acid in Water 2mL of loading solution Washing of interferents •5mL Acetonitrile •5mL 0.2% diethyamine in Acetonitrile Elution (E) 4mL 2% diethylamine in Acetonitrile The elution fraction was filtered and then evaporated under nitrogen and dissolved in the mobile phase before HPLC analysis. Analyse LC-MS/MS

Regulations for Melamine: Codex alimentarius 35th CAC session (july 2012): Maximum limit 0.15mg/kg for liquid infant milk

Catalog number: 6mL-150mg sorbent SCX-25.S.6.150 for cartridges SCX-50.S.6.150 for cartridges Link to AttractSPETM SCX

25 50

AttractSPETM SAX DETERMINATION OF CYANURIC ACID IN MILK PROTOCOL OF PURIFICATION Sample preparation Add 4mL water to 5g liquid infant formula or 1g dry infant formula. Shake during 10-20min with 20mL 50/50 ACN/Water and centrifuge for 10minutes at 3400 rpm. The supernatant is the loading solution Purification with a 6mL/150mg AttractSPETM SAX cartridge Equilibration •5mL 0.1M HCl in Acetonitrile •5mL 0.1M NaOH in Acetonitrile •5mL Acetonitrile •5mL 5% NH4OH in Water Loading 3mL 5% NH4OH in Water 2mL of loading solution Washing of interferents •5mL Acetonitrile Elution (E) 2mL 4% Formic acid in Acetonitrile The elution fraction was filtered and then evaporated under nitrogen and dissolved in the mobile phase before HPLC analysis. Analyse LC-MS/MS 8

Catalog number: 6mL-150mg sorbent SAX-25.S.6.150 for cartridges SAX-50.S.6.150 for cartridges Link to AttractSPETM SAX

25 50

SPE ACCESSORIES POLYINTELL proposes the complete set of equipments required to carry out SPE experiments: Manifold

ACC-MAN1 Like all chromatography techniques, Use of SPE cartridges needs a precise control of flow rate for maintaining reproducible extractions. Solid Phase extraction Vacuum Manifold allows you to control the flow and to process up to 12 (12-port version) or 24 (24-port version) AFFINIMIP® SPE samples simultaneously, to gain significantly time during sample preparation steps.

SPE Adapter & Reservoir kit

ACC-AR1

Tube adapters serve to pile one SPE tube on top of another to provide different selectivities. A larger empty syringe barrel can be stacked on top of a smaller SPE tube to act as a larger load reservoir. Or, they can serve as an adapter for positive pressure methods (e.g. from a syringe or air/ N2 line).

Mini-Vap

ACC-VAP1

The 6-Port Mini-Vap concentrator/evaporator processes six vials at one time. The Mini-Vap includes a needle valve for fine metering of air or nitroge n drying gas.

Mini PUMP

ACC-PUMP

Mini diaphragm vacuum pump for solid phase extraction experiments Portable 5.5L/min ~120 torr vacuum Oil-free portable

Vacuum pump trap

ACC-TRAP

SPE Vacuum pump trap kit Installed between the manifold and the vacuum pump, it collects all liquids that are aspirated preventing contamination of the vacuum pump with a capacity of 1L.

SPE ACCESSORIES – Product list SPE Accessories Manifold SPE Adapter & Reservoir kit

Mini-Vap

Mini PUMP Vacuum pump trap

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Designation

Definition

Reference

SPE Vaccum Manifold

12-port model

ACC-MAN1

SPE Adapter & Reservoir kit

Kit of 12 reservoirs 60ml and adapters for use with 1,3 & 6 mL cartridges

ACC-AR1

Mini Evaporator/Concentrator

6 port Mini-Vap Evaporator/Concentrator for use with 1 to 250mL containers

ACC-VAP1

Mini vacuum pump

Laboport diaphragm vacuum mini pump, 5.5L/min

ACC-PUMP

SPE Vacuum pump trap kit

1L trap kit

ACC-TRAP

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CUSTOM-MADE PRODUCTS & SERVICES POLYINTELL offers full services for the design and synthesis of polymers complying with your specifications. With fully integrated technology platforms in organic, polymer and analytical chemistry, POLYINTELL has been partner in more than 40 projects and helps its customers by innovative solutions for their complexes challenges. POLYINTELL has developed a library of monomers giving a family of selective stationary phases based on its proprietary technology, which have shown a strong potential for the extraction, purification and detection of various compounds.

SPE for analytical applications

Detection kits development

Custommade solutions for:

SPE for radiopharma purifications

Molecularly imprinted polymers development

Did you know ? Molecularly imprinted polymer (MIP) is a polymer with a «memory» of the shape and the functional groups of a target molecule. This material is designed in order to recognize selectively this molecule, even in the presence of compounds with structure and functionality similar to those of this molecule. High molecular recognition properties can be achieved with these MIPs for a variety of molecules and can be useful for its extraction, purification and detection.

Why trusting POLYINTELL? POLYINTELL supplies • The most comprehensive bank of sorbents •From Silica to Polymers •From conventional to very selective •Reversed-phase, ionic exchange, MIPs etc… •Our Expertise on MIP, sample preparation, SPE protocol and detection kit development •Quick and efficient development •Reactivity

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PROJECT DEVELOPMENT No matter if we run a short term project (2-3 days) or long term project ( 4 to 6 months) we always follow a well-established procedure. The following scheme describes an example of a procedure for the development of a custom-made product based on our customer’s requirements. After signing of Confidential Disclosure Agreements (CDA), key data are exchanged. A quotation based on our experience in separation science including a process sheet is developed. During the whole procedure, a permanent feedback is established with you.

Procedure for custom-made polymer phase

Step 1: We offer you a screening of our library which consists of several hundreds POLYINTELL phases to find the suitable one for your separation problem. The knowledge of the structure of the target substance, its functional groups and the solubility data give us valuable hints for the choice of the screening phases.

Feedback and optimisation

Customer

Step 2: For the selected phase, a protocol is implemented for your application. Then samples and / or prototypes are delivered to you for testing, evaluation and validation. Step 3: When the selected phase suits your application and has been validated, a scale-up is planned. A QC report is delivered with the product. The format of the product is correlated to your application and can be bulk material, SPE cartridges, HPLC columns etc…

STEP 1

STEP 2

STEP 3

•Screening

• Elaboration of application protocol • Delivery of samples/ prototypes to customer for validation

• Scale-up • QC report

Bulk material, SPE cartridges or HPLC columns

If you need the development of new polymer for your application, please send us an email to [email protected]l.com or describe your needs using http://www.polyintell.com/services/request-serviceonline/. You can describe your application and our scientists will shortly evaluate your queries before contacting you as soon as possible.

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About POLYINTELL Founded in 2004, POLYINTELL is a worldwide leader in the design and the development of intelligent polymers. Pioneer in Molecularly Imprinted Polymers (MIP) and expert in polymer chemistry, POLYINTELL develops, manufactures and markets innovative solutions for sample preparation, selective extraction, purification and detection of specific target analytes. Our mission is to develop and market innovative products of high value to customers by a practical contribution to their work. By offering you a most comprehensive range of solid phase extraction products, AFFINIMIP® SPE products based on molecularly imprinted polymers, AttractSPETM a range of polymeric phases and Silica based products, associated reagents and small equipment, the analytical chemists can find any solution for sample preparation, selective extraction, sample clean-up and purification needs in various sectors: food safety, pharmaceutical industry, clinical diagnosis, environment and doping. we are your partner of your choice. Furthermore, by exploiting our library of innovative polymers and our know-how in chromatography and solid phase extraction, we have a strong capacity to adapt these polymers to meet any specific requirements and to solve unsatisfied purification and extraction needs.

ORDERING INFORMATION For any order, please, choose one of the following ways:  By mail: POLYINTELL Pharma Parc II Voie de l’innovation 27100 Val de Reuil, FRANCE  By phone: +33 (0)2 32 09 32 70  By fax: +33 (0)2 32 59 61 01  By email: [email protected]  On-line by E-commerce: To save your time and a quicker delivery, order via our web site www.polyintell.com Information about new products or new matrices: Newsletters Stay up-to-date with the news on POLYINTELL's latest innovations by subscribing free of charge to our newsletter, delivering new information straight to your inbox.

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