CORTECS COLUMNS APPLICATIONS NOTEBOOK Table of Contents

+ CORTECS UPLC 1.6 µm Applications Literature Code Page C18 Actinomycins from Streptomyces sp. WA64678 4 Basic Impurities WA64680 5 Beta Blockers WA64672 6 Cat’s Claw Bark (Uncaria Tomentosa) WA64679 7 Chlortalidone Base Degradation WA64673 8 Intact Human Insulin and Five Analogs in Human Plasma WA64695 9 Local Anesthetics WA64675 10 Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) WA64669 11 Omeprazole Tablet Acid Degradation WA64676 12 Sulfa Drugs WA64670 13 Tioconazole Impurities WA64674 14 Tricyclic Antidepressants WA64671 15

CORTECS 2.7 µm Applications

Basic Impurities WA64694 16 Reversed-Phase Quality Control Reference Material (QCRM) WA60706 17

CORTECS UPLC 1.6 µm Applications

C18 Amoxicillin and Clavulanic Acid Oral Suspension WA64690 18 Analgesics WA64682 19 Antibacterials WA64685 20 Astragalus Root WA64692 21 Bradykinin in Human Plasma WA64709 22 Caffeine Metabolites WA64681 23 Catechins and Green Tea WA64684 24 Cephalosporins WA64688 25 Cnidium Monnieri WA64691 26 Cold Medicine Compounds WA64686 27 Erythromycins from Streptomyces sp. WA64693 28

2 Table of Contents

CORTECS UPLC 1.6 µm Applications Continued Literature Code Page C18 Estradiols and Tablet Sample WA64687 29 CONTINUED Phenolics in Echinacea WA64689 30 Statins WA64683 31 Synthetic Cannabinoids from Urine WA64699 32 Synthetic Cannabinoids from Whole Blood WA64700 33

CORTECS 2.7 µm Applications

Abacavir Related Compounds WA64697 34 Antibacterials WA64696 35 LCMS Quality Control Reference Material (QCRM) WA64705 36 Sudan Dyes WA60708 37 Synthetic Cannabinoids WA64698 38

CORTECS UPLC 1.6 µm Applications

HILIC Acetylcholine, Histamine, and their Metabolites in Human CSF WA64702 39 Acyclovir and Guanine WA64666 40 Cold Medicine Compounds WA64667 41 Histidine Dipeptides WA64668 42 Local Anesthetics WA64664 43 Morphine Metabolites WA64662 44 Nucleic Acid Bases WA64663 45 Paraquat and Diquat in Drinking Water WA64703 46 Paraquat and Diquat in Potato and Wheat WA64710 47 Water Soluble Vitamins WA64665 48

CORTECS 2.7 µm Applications

Basic Drugs in River Water WA60707 49 HILIC Quality Control Reference Material (QCRM) WA64704 50 Morphine Metabolites WA64701 51

3 [ C18+ APPLICATION HIGHLIGHT ] Analysis of Actinomycins from Streptomyces sp. using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® with Xevo® G2 QToF Mass Spectrometer

® ® Column: CORTECS UPLC C18+, 1.6 µm, 2.1 x 50 mm (p/n 186007114) Mobile phase A: Water with 0.1% formic acid O H O Mobile phase B: Acetonitrile with 0.1% formic acid N O N Flow rate: 0.6 mL/min N NH O O Gradient: 2 to 98% B in 5.5 minutes, hold 1 minute, O O H N N equilibrate at 2% B HN O O N H H O Run time: 7 minutes H2N N O N O N O O O Injection volume: 1 µL O Column temp.: 30 °C Actinomycin D Scan mode: ESI+ 200–1500 amu mw: 1255.42 C62H86N12O16 Cone voltage: 30 V Desolvation gas: 800 L/hr Desolvation temp.: 280 °C Capillary voltage: 3 kV Sample: 10 mL Streptomyces sp. fermentation broth extracted with 10 mL 80:20 EtoAc/methanol Identification: Compounds identified by accurate mass using MassLynx® Software

Actinomycins from Streptomyces sp. 3.88 1: TOF MS ES+ BPI 8.26e4

Compounds: 1. Actinomycin analog, m/z 1327 4 2. Actinomycin analog, m/z 1305 3. Actinomycin analog, m/z 1257 4. Actinomycin D

% 5. Actinomycin analog, m/z 1269 6. Actinomycin analog, m/z 1277 7. Actinomycin analog, m/z 1255

2 3 3.12 3.33

1 5 5.79 6.74 2.96 4.15 7 6.29 6 4.48 1.90 6.08

0 1 2 3 4 5 6 Min

©2014 Waters Corporation. Waters, The Science of What’s Possible, CORTECS, ACQUITY UPLC, Xevo, UPLC, and MassLynx are registered trademarks of Waters Corporation. WA64678 4 [ C18+ APPLICATION HIGHLIGHT ]

Analysis of Basic Impurities using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® I-Class with ACQUITY® PDA

® ® Columns: CORTECS UPLC C18+, 1.6 µm, 2.1 x 50 mm (p/n 186007114)

Competitor Solid-Core C18, 1.7 µm, 2.1 x 50 mm Mobile phase A: 0.1% formic acid in water Mobile phase B: 0.1% formic acid in acetonitrile N Detection: UV at 254 nm N Flow rate: 0.6 mL/min Imipramine Gradient: See table Injection volume: 5.0 µL Column temp.: 30 °C Sample preparation: Imipramine (0.5 mg/mL), with various concentrations of amitriptyline (0.5, 2.5, 5, Time Flow Rate (min) (mL/min) %A %B and 10%) prepared in water 0.0 0.6 75 25 2.0 0.6 65 35 3.0 0.6 5 95 3.1 0.6 75 25 5.0 0.6 75 25

Spiked impurity analysis

0.20

0.16 Spiked impurity 0.5%, 2.5%, 5%, 10% 0.12 CORTECS UPLC C18+

AU 1.6 µm, 2.1 x 50 mm 0.08

0.04 Imipramine 0.00

0.20 Competitor Solid-Core C18 0.16 1.7 µm, 2.1 x 50 mm

Spiked impurity 0.12 0.5%, 2.5%, 5%, 10% AU 0.08 Imipramine Comparative separations may not be representative 0.04 in all applications. 0.00

0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 Min

Analysis of Beta Blockers using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® I-Class with ACQUITY® PDA

® ® Column: CORTECS UPLC C18+, 1.6 µm, 2.1 x 50 mm (p/n 186007114) Mobile phase A: 0.1% formic acid in water H N O OH Mobile phase B: 0.1% formic acid in acetonitrile H N OH H H2N 2D UV channels: 260 nm O N HO Flow rate: 0.4 mL/min Pindolol Labetolol Gradient: See table OH H Sampling rate: 80 pts/sec O N CH3 O N O H OH CH3 Time constant filter: Fast H3C N CH3 H O Injection volume: 1.0 µL Acebutolol Propranolol Column temp.: 30 °C

OH Sample diluent: Water H O N

Time Flow Rate O (min) (mL/min) %A %B Oxprenolol 0.0 0.5 95 5 4.5 0.5 5 95 5.0 0.5 5 95 5.1 0.5 95 5 6.0 0.5 95 5

Beta Blockers Compounds: 1. Pindolol (10 µg/mL) 0.10 1 2 2. Acebutolol (10 µg/mL) 3. Oxprenolol (20 µg/mL) 4. Labetolol (30 µg/mL) 0.08 5. Propranolol (10 µg/mL)

0.06 AU

0.04

5 0.02 3

0.00

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 Min

Analysis of Cat’s Claw Bark using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® with Xevo® G2 QToF Mass Spectrometer

® ® Column: CORTECS UPLC C18+, 1.6 µm, 2.1 x 100 mm (p/n 186007116) Mobile phase A: Water with 0.1% formic acid

Mobile phase B: Acetonitrile with 0.1% formic acid N O

Flow rate: 0.6 mL/min O N O O Gradient: 15 to 30% B in 4.5 minutes, ramp to 90% B in 1 minute, H hold to 7 minutes, equilibrate at 15% B Rhynchophylline C22H28N2O4 Run time: 8 minutes mw 384.47 Injection volume: 1 µL O Column temp.: 30 °C N O

Scan mode: ESI+ 200–1000 amu O N O O Cone voltage: 30 V H Rhynchophylline-N-oxide Desolvation gas: 800 L/hr C22H28N2O5 mw 400.47 Desolvation temp.: 280 °C Capillary voltage: 3 kV Sample: 485 mg Cat’s Claw Bark (Uncaria tomentosa) extracted with 4 mL 50:50 EtoAc/methanol Identification: Mass extracted from total ion chromatogram (TIC) and compounds identified by accurate mass using MassLynx® Software

Cat’s Claw Bark 100 6.49 1: TOF MS ES+ BPI 1.18e5 1.53 2.12 6.74 1.49 Cat’s claw extract % 1.61 2.55 6.99

2.12 100 1: TOF MS ES+ 385.212 9.17e4 Rhynchophylline and isomer % 1.90

2.12 98 1: TOF MS ES+ 401.204 1.97e3 Rhynchophylline N-oxide % 6.43

-2

1 2 3 4 5 6 7 Min

©2014 Waters Corporation. Waters, The Science of What’s Possible, CORTECS, ACQUITY UPLC, Xevo, UPLC, and MassLynx are registered trademarks of Waters Corporation. WA64679 7 [ C18+ APPLICATION HIGHLIGHT ] Analysis of Chlortalidone Base Degradation using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® I-Class with ACQUITY® PDA

® ® Column: CORTECS UPLC C18+, 1.6 µm, 2.1 x 50 mm (p/n 186007114) Mobile phase A: 0.1% formic acid in water Cl OH Mobile phase B: 0.1% formic acid in acetonitrile H2N S Detection: UV at 254 nm OOHN Gradient: See table O Chlortalidone Injection volume: 1.0 µL

Column temp.: 30 °C CH3OSO22NH Sample diluent: Methanol Cl NH

Sample preparation: Chlortalidone subjected to 0.2 mL 1 N sodium O hydroxide and heated at 60 °C for 20 hours Chlortalidone Degradation Product: 2-Chloro-5-(1-methoxy3-oxo-1-isoindoline)- benzene sulfonamide Time Flow Rate (min) (mL/min) %A %B Initial 0.6 95 5 5.0 0.6 5 95 5.5 0.6 5 95 5.6 0.6 95 5 7.0 0.6 95 5

Chlortalidone Base Degradation Compounds: 1. Chlortalidone degradation product, unidentified 2 3 2. Chlortalidone 3. 2-Chloro-5-(1-methoxy3-oxo-1-isoindoline)- benzene sulfonamide 0.60

0.40 AU

0.20 1

0.00

0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 Min

©2014 Waters Corporation. Waters, The Science of What’s Possible, CORTECS, ACQUITY UPLC, ACQUITY, and UPLC are registered trademarks of Waters Corporation. WA64673 8 [ C18+ APPLICATION HIGHLIGHT ] Analysis of Intact Human Insulin and Five Analogs in Human Plasma using CORTECS UPLC 1.6 µm Columns

CONDITIONS Human Insulin A chain insulin System: ACQUITY UPLC® I-Class with 2D Technology,

configured for at-column dilution with trap Insulin B chain and back elution with Xevo® TQ-S Mass Spectrometer

® ® Humalog Insulin A chain Analytical column: CORTECS UPLC C18+, 1.6 µm, (insulin lispro) 2.1 x 50 mm (p/n 186007114)

® Insulin B chain Trap column: XBridge C18 IS™, 3.5 µm, 2.1 x 20 mm (p/n 186003019) Lantus Insulin A chain (insulin glargine) Elution mobile phase A: 0.1% formic acid in water

Elution mobile phase B: 0.1% formic acid in acetonitrile Insulin B chain Gradient: Load for two minutes; switch valve and back elute Nova log Insulin A chain from trap column onto analytical column with a (insulin apart) linear gradient from 15 to 40% B over four minutes Insulin B chain Elution flow rate: 0.25 mL/min

Levemir Insulin A chain Column temp.: 60 °C (insulin detemir) Sample temp.: 15 °C Insulin B chain Injection volume: 30 µL

Collection plates: Waters® 1-mL ACQUITY® collection plates Apidra Insulin A chain (insulin glulisine)

To see the full application note, visit www.waters.com Insulin B chain and search for literature code: 720004727EN

Human Insulin, five-insulin analogs, and bovine insulin

5.52 4.28 100 100 Insulin glulisine Insulin detemir 3.60 % RT 4.28 min RT 5.52 min

% 5.81 0 2468

0 2468 4.13 100 Insulin glargine RT 4.13 min

4.29 % 100 5.73 5.28 0 Insulin glulisine 2468

% RT 4.29 min 4.27 5.26 100 Insulin aspart 0 RT 4.27 min

2468 % 4.04 5.78 0 4.30 2468 100 3.60 Human insulin 4.23 RT 4.30 min 100 Bovine insulin (IS) % RT 4.23 min % 5.26 5.46 3.21 5.19 0 0 2468 Min. 2 468 Min.

©2014 Waters Corporation. Waters, The Science of What’s Possible, XBridge, CORTECS, ACQUITY UPLC, ACQUITY, Xevo, and UPLC are registered trademarks of Waters Corporation. IS is a trademark of Waters Corporation. WA64695 9 [ C18+ APPLICATION HIGHLIGHT ] Analysis of Local Anesthetics by Reversed-Phase LC using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® I-Class with ACQUITY® PDA

® ® Column: CORTECS UPLC C18+, 1.6 µm, 2.1 x 50 mm (p/n 186007114) Mobile phase A: 0.1% formic acid in water H O Mobile phase B: 0.1% formic acid in acetonitrile N N N N H Detection: UV at 245 nm O Gradient: See table Lidocaine Bupivacaine Flow rate: 0.6 mL/min O O Injection volume: 1.0 µL N O N O

Column temp.: 30 °C H N N 2 H

Sample diluent: Water Butacaine Tetracaine

Time Flow Rate N O (min) (mL/min) %A %B O Initial 0.6 95 5 N O O N 1.0 0.6 65 35 H H2N

2.0 0.6 65 35 Dibucaine Benzocaine 5.5 0.6 5 95 5.6 0.6 95 5 7.0 0.6 95 5

Local Anesthetics Compounds: 1. Lidocaine (50 µg/mL) 2. Bupivacaine (50 µg/mL) 3. Butacaine (50 µg/mL) 0.10 5 4. Tetracaine (50 µg/mL) 5. Dibucaine (10 µg/mL) 6. Benzocaine (10 µg/mL) 0.08

0.06 AU 3

0.04 4 1

2 6 0.02

0.00

0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Min

Analysis of NSAIDs using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® I-Class with ACQUITY® PDA

® ® Column: CORTECS UPLC C18+, 1.6 µm, 2.1 x 50 mm (p/n 186007114) Mobile phase A: 0.1% formic acid in water O Mobile phase B: 0.1% formic acid in acetonitrile N O COOH COOH Detection: UV at 270 nm Data rate 20 pts/sec Tolmetin Fenoprofen Filter time: Normal O Cl Gradient: See table COOH N

Injection volume: 1.0 µL H CO 3 H3CO Column temp.: 30 °C COOH Naproxen Indomethacin Sample: NSAIDs mix at 50–100 µg/mL in water

COOH Cl H Time Flow Rate N (min) (mL/min) %A %B Cl Initial 0.8 65 35 Diclofenac 2.2 0.8 35 65 2.3 0.8 35 65 2.4 0.8 65 35 3.0 0.8 65 35

NSAIDs Compounds: 1. Tolmetin 2. Naproxen 3. Fenoprofen 4. Indomethacin 5. Diclofenac 1 5 2 0.25

0.20

0.15 AU

0.10 4 3 0.05

0.00

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 Min

©2014 Waters Corporation. Waters, The Science of What’s Possible, CORTECS, UPLC, ACQUITY UPLC, and ACQUITY are registered trademarks of Waters Corporation. WA64669 11 [ C18+ APPLICATION HIGHLIGHT ] Analysis of Omeprazole Tablet Acid Degradation using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® I-Class with ACQUITY® PDA

® ® Column: CORTECS UPLC C18+, 1.6 µm, 2.1 x 50 mm (p/n 186007114) Mobile phase A: 0.1% formic acid in water H Mobile phase B: 0.1% formic acid in acetonitrile N O N S N OCH3 Detection: UV at 254 nm H3CO Gradient: See table Omeprazole

Flow rate: 0.6 mL/min H N N Injection volume: 1.0 µL S N OCH3 H3CO Column temp.: 30 °C Omeprazole sulfide Sample diluent: 50% methanol in water Sample preparation: Omeprazole subjected to 1 N HCl and left at room temperature for 20 minutes

Time Flow Rate (min) (mL/min) %A %B Initial 0.6 95 5 5.0 0.6 5 95 5.5 0.6 5 95 5.6 0.6 95 5 7.0 0.6 95 5

Omeprazole Acid Degradation Compounds: 1. Omeprazole 1.00 1 2. Omeprazole sulfide

0.80

0.60 AU 0.40 Standard

0.20

0.00

2 0.30

0.20 AU Acid Degradation

0.10

0.00

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 Min

Analysis of Sulfa Drugs using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® H-Class with ACQUITY® PDA

® ® Column: CORTECS UPLC C18+, 1.6 µm, 2.1 x 50 mm (p/n 186007114) Mobile phase A: 0.1% formic acid in water O O S O O N Mobile phase B: 0.1% formic acid in acetonitrile S S N N N N H H Detection: UV at 254 nm H2N H2N

Flow rate: 0.5 mL/min Sulfathiazole Sulfamerazine Gradient: See table H O O N O N N S N Injection volume: 4.0 µL S N N O H O Column temp.: 30 °C H2N H2N

Sample diluent: Water Sulfamethazine Sulfamethoxypyridazine

N Cl N O N O O O O S Time Flow Rate S N N H (min) (mL/min) %A %B H H2N H2N 0.0 0.5 95 5 Sulfachloropyridazine Sulfamethoxazole 5.0 0.5 40 60

5.5 0.5 40 60 O O O N S N 5.6 0.5 95 5 H 7.0 0.5 95 5 H2N Sulfasoxazole

Sulfa Drugs Compounds: 1. Sulfathiazole (10 µg/mL) 2. Sulfamerazine (10 µg/mL) 1.40 7 3. Sulfamethazine (10 µg/mL) 4. Sulfamethoxypyridazine (10 µg/mL) 1.20 5. Sulfachloropyridazine (10 µg/mL) 6. Sulfamethoxazole (10 µg/mL) 7. Sulfasoxazole (10 µg/mL) 1.00

0.80 AU

0.60

3 4 0.40 2 1 5 6 0.20

0.00

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Min

Analysis of Tioconazole Impurities using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® with ACQUITY® PDA

® ® Column: CORTECS UPLC C18+, 1.6 µm, 2.1 x 50 mm (p/n 186007114) Mobile phase A: 0.1% formic acid in water N N Mobile phase B: 0.1% formic acid in acetonitrile Cl Cl N N Detection: UV at 219 nm S S O Cl O Cl Gradient: See table Cl Flow rate: 0.6 mL/min Tioconazole Related Compound A Injection volume: 4.0 µL N N Cl Cl Column temp.: 30 °C N N Cl Br Sample diluent: Methanol S S O Cl O Cl Sample: Tioconazole USP Related Compounds Cl Cl

Related Compound B Related Compound C Time Flow Rate (min) (mL/min) %A %B 0.0 0.6 98 2 4.0 0.6 25 75 4.5 0.6 25 75 4.6 0.6 98 2 6.0 0.6 98 2

Tioconazole Impurities Compounds: 1. Related Compound A (10 µg/mL) 2. Tioconazole (20 µg/mL) 0.40 3. Related Compound B (10 µg/mL) 2 4. Related Compound C (10 µg/mL)

0.30

0.20 1 3 AU

4 USP Resolution3,4: 1.7

0.10

0.00

1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Min

Analysis of Tricyclic Antidepressants using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® H-Class with ACQUITY® PDA

® ® Column: CORTECS UPLC C18+, 1.6 µm, 2.1 x 50 mm (p/n 186007114) Mobile phase A: 0.1% formic acid in water O Mobile phase B: 0.1% formic acid in acetonitrile

Detection: UV at 254 nm N N H H Flow rate: 0.6 mL/min Nordoxepin Nortriptyline Gradient: See table Injection volume: 1.0 µL

Column temp.: 40 °C N N N Sample diluent: Water H

Trimipramine Protriptyline

Time Flow Rate (min) (mL/min) %A %B 0.0 0.6 72 28 3.0 0.6 65 35 N

3.5 0.6 5 95 Amitriptyline 3.6 0.6 72 28 6.0 0.6 72 28

Tricyclic Antidepressants Compounds: 1. Nordoxepin (10 µg/mL) 2. Protriptyline (10 µg/mL) 3. Nortriptyline (10 µg/mL) 4. Amitriptyline (10 µg/mL) 1 0.040 5. Trimipramine (10 µg/mL)

4 3 0.030 5

0.020 AU 2

0.010

0.000

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Min

Analysis of Basic Impurities using CORTECS 2.7 µm Columns

CONDITIONS System: Alliance® HPLC with 2998 Photodiode Array Detector

® Column: CORTECS C18+, 2.7 µm, 4.6 x 150 mm (p/n 186007408) Mobile phase A: 0.1% formic acid in water Mobile phase B: 0.1% formic acid in acetonitrile Detection: UV at 254 nm N Flow rate: 1.5 mL/min N Gradient: See table Imipramine Injection volume: 36 µL Column temp.: 30 ˚C Sample preparation: Imipramine (0.5 mg/mL), with various concentrations of amitriptyline (10 µg/mL, 5 µg/mL, and 0.5 µg/mL) prepared in water

Time (min) %A %B Curve 0.0 75 25 – 11.3 65 35 6 12.0 65 35 6 12.1 75 25 6 15.0 75 25 6

Imipramine spiked impurity analysis

0.10

0.08 Pressuremax: 3750 psi

0.06 Spiked Impurity ne mi AU ra

ip 10 µg/mL 0.04 Im

5 µg/mL 0.02

0.5 µg/mL

0.00

2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 Min

©2014 Waters Corporation. Waters, The Science of What’s Possible, CORTECS, and Alliance are registered trademarks of Waters Corporation. WA64694 16 [ C18+ APPLICATION HIGHLIGHT ] Analysis of the Reversed-Phase Quality Control Reference Material (QCRM) using CORTECS 2.7 µm Columns

CONDITIONS System: ACQUITY UPLC® with PDA Detector O ® O N Column: CORTECS C18+, 2.7 µm, HN H OH 2.1 x 100 mm (p/n 186007397) O N H Mobile phase A: 0.1% formic acid in water Uracil Propranolol Acenaphthene Mobile phase B: 0.1% formic acid in acetonitrile

Flow rate: 1.0 mL/min O Gradient: See table O HO N Injection volume: 2.0 µL Column temp.: 30 ˚C Butylparaben Amitriptyline Naphthalene Sample: Reversed-Phase QCRM (p/n 186006363) O

O O

Time Flow Rate O (min) (mL/min) %A %B Curve Initial 1.0 95 5 – Dipropylphthalate 2.70 1.0 5 95 6 3.00 1.0 5 95 6 3.12 1.0 95 5 6 4.00 1.0 95 5 6

Reversed-Phase QCRM Compounds: 1. Uracil 2. Propranolol 3 4 3. Amitriptyline 6 3.0e-1 4. Butylparaben 5. Naphthalene 6. Dipropylphthalate 2.5e1 7. Acenaphthene 7

2.0e-1 1 2 AU 1.5e-1

1.0e-1 5 Solvent peak

5.0e-2

0.0

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 Min.

©2014 Waters Corporation. Waters, The Science of What’s Possible, CORTECS, and ACQUITY UPLC are registered trademarks of Waters Corporation. WA60706 17 [ C18 APPLICATION HIGHLIGHT ] Analysis of Amoxicillin and Clavulanic Acid Oral Suspension using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® with ACQUITY® PDA

® ® Column: CORTECS UPLC C18, 1.6 µm, 2.1 x 100 mm (p/n 186007095) Separation mode: Isocratic (92:8 mobile phase A:B) HO Mobile phase A: 20 mM potassium phosphate buffer pH 5.2 O Mobile phase B: 85:15 mobile phase A/acetonitrile H H H2N HN S Detection: UV at 230 nm N O OH Flow rate: 0.4 mL/min O Amoxicillin Injection volume: 2.0 µL

Column temp.: 30 °C H H H2N S Sample diluent: Water N O OH Standards: Prepared to listed concentrations O 6-Aminopenicillanic acid Sample preparation: Amoxicillin suspension diluted with water.

Sample filtered through a 0.2 µm filter. H O OH

N OH O H O Clavulanic acid

Amoxicillin and Clavulanic Acid Oral Suspension Compounds: 1. Clavulanic acid (100 µg/mL) 2. 6-APA (50 µg/mL) 3 3. Amoxicillin (100 µg/mL) 0.20

0.15 Suspension Sample

AU 0.10

0.05 1

0.00

0.30

AU 0.20 Standards 0.10 1 2

0.00

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Min

Analysis of Analgesics using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® I-Class with ACQUITY® PDA

® ® Column: CORTECS UPLC C18, 1.6 µm, 2.1 x 50 mm (p/n 186007093) Mobile phase A: 0.1% formic acid in water HO O Mobile phase B: 0.1% formic acid in acetonitrile O N CH3 N H Detection: UV at 260 nm H OH Gradient: See table Acetaminophen 2-Acetamidophenol Flow rate: 0.6 mL/min O O Injection volume: 1.0 µL N N O O N N Column temp.: 30 °C OOH Sample diluent: Water Acetylsalicylic acid Caffeine

Time Flow Rate O (min) (mL/min) %A %B N H Initial 0.6 90 10 Acetanilide 0.5 0.6 90 10 1.2 0.6 50 50 1.5 0.6 90 10 3.0 0.6 90 10

Analgesics Compounds: 1. Acetaminophen (0.1 mg/mL) 2. Caffeine (0.1 mg/mL) 3. 2-Acetamidophenol (0.1 mg/mL) 4 4. Acetanilide (0.1 mg/mL) 1 5. Acetylsalicylic acid (0.1 mg/mL) 0.20

0.15

AU 0.10

0.05 3 5

0.00

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Min

Analysis of Antibacterials using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® I-Class with ACQUITY® PDA

® ® Column: CORTECS UPLC C18, 1.6 µm, 2.1 x 50 mm (p/n 186007093)

Competitor Solid-Core C18, 1.7 µm, 2.1 x 50 mm

Mobile phase A: 0.1% formic acid in water O H NH2 N N H Mobile phase B: 0.1% formic acid in acetonitile N N N O S N O O N Detection: UV at 254 nm O Flow rate: 0.6 mL/min Carbadox Sulfaquinoxaline Gradient: See table Injection volume: 1.0 µL O N N N Column temp.: 30 °C O COOH COOH O Sample diluent: 20% acetonitrile in water O Oxolinic acid Nalidixic acid

Time Flow Rate (min) (mL/min) %A %B Initial 0.6 95 5 4.0 0.6 50 50 4.1 0.6 95 5 5.0 0.6 95 5

Antibacterials Compounds: 0.70 1. Carbadox (0.1 mg/mL) 0.60 1 3 2. Oxolinic acid (0.1 mg/mL)

0.50 3. Sulfaquinoxaline (0.1 mg/mL) 4. Nalidixic acid (0.1 mg/mL) 0.40

AU 4 0.30 Cortecs UPLC C18 1.6 µm, 2.1 x 50 mm 0.20 2 0.10

0.00

0.70

0.60 1 3 0.50

0.40

AU Competitor Solid-Core C18 0.30 1.7 µm, 2.1 x 50 mm 0.20 4 Comparative separations may not be representative 0.10 in all applications. 2 0.00

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Min

Analysis of Astragalus Root using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® with Xevo® G2 QToF Mass Spectrometer

® ® Column: CORTECS UPLC C18, 1.6 µm, 2.1 x 100 mm (p/n 186007095) HO Mobile phase A: Water with 0.1% formic acid O Mobile phase B: Acetonitrile with 0.1% formic acid HO O HO O O H H OH Flow rate: 0.6 mL/min HO OH

HO Gradient: 2 to 98% B in 5.5 minutes O O OH Run time: 7 minutes Ononin Cycloastragenol C22H22O9 C30H50O5 Injection volume: 1 µL 430.40 490.72 Column temp.: 30 °C Scan mode: ESI+ 200–1000 amu HO O HO O OH

Cone voltage: 30 V O O O O Desolvation gas: 800 L/hr Calycosin Formononetin C16H12O5 C16H12O4 Desolvation temp.: 280 °C 284.26 268.26 Capillary voltage: 3 kV Sample: 375 mg Astragalus root extracted with 4 mL 50:50 EtoAc/methanol Identification: Mass extracted from total ion chromatogram (TIC) and compounds identified by accurate mass using MassLynx® Software

4.42 Astragalus Root 2.35 4.21 1: TOF MS ES+ Astragalus extract 5.28 BPI 2.03 2.10e5 % 5.59 6.31 0

2.55 99 1: TOF MS ES+ Ononin 431.134

% 5.91e4

-1

99 3.18 1: TOF MS ES+ Cycloastragenol 491.372 % 3.37 2.39e3 -1

2.55 99 1: TOF MS ES+ 3.26 Calycosin 269.083

% 1.30e3

-1

2.76 98 1: TOF MS ES+ Formonenetin 285.076 2.18e3 % 2.02 -2 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 Min

©2014 Waters Corporation. Waters, The Science of What’s Possible, CORTECS, ACQUITY UPLC, Xevo, UPLC, and MassLynx are registered trademarks of Waters Corporation. WA64692 21 [ C18 APPLICATION HIGHLIGHT ] Quantitative SPE LC-MS/MS Assay for Bradykinin in Human Plasma using CORTECS UPLC 1.6 µm Column

CONDITIONS System: ACQUITY UPLC® with a Xevo® TQ-S Mass Spectrometer

® ® Column: CORTECS UPLC C18, 1.6 µm, 2.1 mm x 50 mm Mobile phase A: 0.1% formic acid in water O N O O OH Mobile phase B: 0.1% formic acid in acetonitrile HN2 H N N N O N N N H H H COOH Gradient: see Table O O N O H O Column temp.: 35 °C NH NH

HN NH2 N NH2 Sample temp.: 15 °C H Bradykinin Amino Acid Sequence: RPPGFSPFR Inj. volume: 10 µL Total run time: 3.5 minutes Time Flow Rate Collection plates: Waters 1 mL ACQUITY® (min) (mL/min) %A %B Curve Collection Plates 0.00 0.400 95.0 5.0 Initial Ionization mode: ESI positive 0.15 0.400 95.0 5.0 6 1.75 0.400 25.0 75.0 6 Capillary voltage: 3.0 kV 2.00 0.400 5.0 95.0 6 Desolvation temp.: 500 °C 2.50 0.400 5.0 95.0 6 Cone gas flow: 150 L/hr 2.60 0.400 95.0 5.0 6 Desolvation gas flow: 1000 L/hr 3.00 0.400 95.0 5.0 6

Endogenous levels of bradykinin extracted from human plasma

Area

A Plasma Donor #1 EDTA With Protease Inhibitor 90 pg/mL Intensity [Counts] Intensity

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 Min.

Plasma Donor #1 EDTA B No Protease Inhibitor 250 pg/mL Intensity [Counts] Intensity 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 Min.

Plasma Donor #2 EDTA No Protease Inhibitor C Unknown Sample Collection and Protocol 4,300 pg/mL Intensity [Counts] Intensity 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 Min. Plasma Donor #3 EDTA No Protease Inhibitor Unknown Sample Collection and Protocol D 3,100 pg/mL Intensity [Counts] Intensity 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 Min.

©2014 Waters Corporation. Waters, The Science of What’s Possible, ACQUITY UPLC, Xevo, CORTECS, UPLC, ACQUITY, and Oasis are registered trademarks of Waters Corporation. WA64709 22 [ C18 APPLICATION HIGHLIGHT ]

Analysis of Caffeine Metabolites using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® I-Class with ACQUITY® PDA

® ® Column: CORTECS UPLC C18, 1.6 µm, 2.1 x 50 mm (p/n 186007093) Mobile phase A: 0.1% formic acid in water O O Mobile phase B: 0.1% formic acid in acetonitrile H N N N N O Detection: UV at 280 nm O N N O N N H H H Gradient: See table 1-Methylxanthine 1,7-Dimethyluric acid Flow rate: 0.43 mL/min O O Injection volume: 2.0 µL N N HN N

Column temp.: 30 °C O N N O N N

Sample diluent: Water Caffeine Theobromine

O Time Flow Rate N (min) (mL/min) %A %B N O N N Initial 0.6 95 5 H 2.2 0.6 76 24 1,7-Dimethylxanthine 2.5 0.6 95 5 4.0 0.6 95 5

Caffeine Metabolites Compounds: 1. 1-Methylxanthine (0.1 mg/mL) 2. Theobromine (0.1 mg/mL) 2 3. 1,7-Dimethyluric acid (0.1 mg/mL) 0.40 3 4. 1,7-Dimethylxanthine (0.1 mg/mL) 5. Caffeine (0.1 mg/mL) 5

0.30

AU 0.20

0.10

0.00

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 Min

Analysis of Catechins and Green Tea using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® H-Class with ACQUITY® PDA

® ® Column: CORTECS UPLC C18, 1.6 µm, 2.1 x 100 mm (p/n 186007095) Separation mode: Isocratic

Mobile phase: 90:10 water/acetonitrile with 0.1% formic acid OH OH OH Detection: UV at 215 nm HO O HO O OH OH Flow rate: 0.53 mL/min OH OH OH Injection volume: 1.4 µL OH Column temp.: 30 °C Epigallocatechin Epicatechin Standards: Prepared to 0.1 mg/mL in water OH Sample: 0.2 g green tea extracted with OH OH HO O 10 mL methanol and diluted using OH HO O ® OH Milli-Q water O OH OH OH O OH OH OH Catechin Gallocatechin gallate

Catechins Compounds: 1. Epigallocatechin 2 2. Catechin 3 3. Epicatechin

0.40 1 4. Gallocatechin gallaete

Standard Mix

AU 0.20 4

0.00

0.30

AU 0.20 Green Tea 4 0.10 3

0.00

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 Min

©2014 Waters Corporation. Waters, The Science of What’s Possible, CORTECS, ACQUITY UPLC, ACQUITY, and UPLC are registered trademarks of Waters Corporation. All other trademarks are property of their respective owners. WA64684 24 [ C18 APPLICATION HIGHLIGHT ]

Analysis of Cephalosporins using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® I-Class with ACQUITY® PDA

® ® Column: CORTECS UPLC C18, 1.6 µm, 2.1 x 50 mm (p/n 186007093) Mobile phase A: 0.1% formic acid in water HO O O Mobile phase B: 0.1% formic acid in acetonitrile H H H H S S N N H H Detection: UV at 254 nm NH2 N NH2 N O O Cl Flow rate: 0.5 mL/min O OH O OH Gradient: See table Cefadroxil Cefaclor Injection volume: 1.4 µL

O O Column temp.: 30 °C H H H H S S N N H H Sample diluent: Water NH2 N NH2 N O O O OH O OH

Time Flow Rate Cephalexin Cephradine (min) (mL/min) %A %B Initial 0.5 95 5 2.0 0.5 80 20 2.1 0.5 95 5 3.0 0.5 95 5

Cephalosporins Compounds: 1. Cefadroxil (20 µg/mL) 0.120 2. Cefaclor (20 µg/mL) 3. Cephalexin (20 µg/mL) 0.110 4. Cephradine (50 µg/mL) 0.100

0.090

1 0.080 2 3 0.070

0.060 AU 4

0.050

0.040

0.030

0.020

0.010

0.000

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 Min

Analysis of Cnidium Monnieri using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® with Xevo® G2 QToF Mass Spectrometer

® ® Column: CORTECS UPLC C18, 1.6 µm, 2.1 x 50 mm (p/n 186007093) Mobile phase A: Water with 0.1% formic acid Mobile phase B: Acetonitrile with 0.1% formic acid OOO Flow rate: 0.6 mL/min Gradient: 2 to 98% B in 5.5 minutes, Osthol hold 1 minute, equilibrate at 2% B mw 244.28 C H O Run time: 7 minutes 15 16 3 Injection volume: 1 µL Column temp.: 30 °C Scan mode: ESI+ 200–500 amu Cone voltage: 30 V Desolvation gas: 800 L/hr Desolvation temp.: 280 °C Capillary voltage: 3 kV Sample: 600 mg Cnidium Monnieri extracted with 8 mL 50:50 EtoAc/methanol Identification: Mass extracted from total ion chromatogram (TIC) and compounds identified by accurate mass using MassLynx® Software

Cnidium Monnieri

1.68 2.95 1: TOF MS ES+ BPI 2.82 9.82e3 Extract 3.90

0.27 1.77 % 1.43 4.07 4.32 2.16 2.65 3.12 3.51

2.95 100 1: TOF MS ES+ 245.119 Osthol 1.21e3 %

-0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Min

©2014 Waters Corporation. Waters, The Science of What’s Possible, CORTECS, ACQUITY UPLC, Xevo, UPLC, and MassLynx are registered trademarks of Waters Corporation. WA64691 26 [ C18 APPLICATION HIGHLIGHT ] Analysis of Cold Medicine Compounds by Reversed-Phase LC using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® H-Class with ACQUITY® PDA

® ® Column: CORTECS UPLC C18, 1.6 µm, 2.1 x 100 mm (p/n 186007095) Mobile phase A: 0.1% trifluoroacetic acid in water

H3CO Mobile phase B: Acetonitrile HO O H Detection: UV at 265 nm N H NCH3 Flow rate: 0.6 mL/min Acetaminophen Dextromethorphan Gradient: See table Injection volume: 1.0 µL Cl N N O Column temp.: 30 °C N Standards: Prepared to listed concentrations in water Sample: 1 mL cough syrup diluted to 10 mL with water Chlorpheniramine Diphenhydramine

Time Flow Rate OH (min) (mL/min) %A %B H N Initial 0.5 95 5 0.5 0.5 95 5 Pseudoephedrine 4.0 0.5 52 48 4.5 0.5 52 48 4.6 0.5 95 5 6.0 0.5 95 5

Cold Medicine Compounds Compounds: 1. Acetaminophen (10 µg/mL)

0.050 3 2. Pseudoephedrine (200 µg/mL) Standards 3. Chlorpheniramine (100 µg/mL) 0.040 4. Dextromethorphan (100 µg/mL) 5. Diphenhydramine (100 µg/mL) 0.030

AU 4 0.020 1 2 5 0.010

0.000

1 2.00 Cough Syrup 1.50 AU 1.00

3 0.50 Phenylephrine Doxylamine

0.00

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 Min

©2014 Waters Corporation. Waters, The Science of What’s Possible, CORTECS, ACQUITY UPLC, ACQUITY, and UPLC are registered trademarks of Waters Corporation. WA64686 27 [ C18 APPLICATION HIGHLIGHT ] Analysis of Erythromycins from Streptomyces sp. using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® with Xevo® G2 QToF Mass Spectrometer

® ® Column: CORTECS UPLC C18, 1.6 µm, 2.1 x 100 mm (p/n 186007095) Mobile phase A: Water with 0.1% formic acid OH HO OH Mobile phase B: Acetonitrile with 0.1% formic acid O O Flow rate: 0.6 mL/min O O O O OH O O Gradient: 2 to 98% B in 5.5 minutes, O O N O O N hold 1 minute, equilibrate at 2% B OH OH O OH O HO Run time: 7 minutes O O OH Injection volume: 1 µL Erythromycin A Erythromycin A enol ether Column temp.: 30 °C mw 733.93 mw 715.91 C37H67NO13 C37H65NO12 Scan mode: ESI+ 200–1500 amu

Cone voltage: 30 V OH Erythromycin B mw 717.93 O Desolvation gas: 800 L/hr O C37H67NO12 O O Desolvation temp.: 280 °C OH O O N Capillary voltage: 3 kV OH OH O Sample: 10 mL Streptomyces sp. fermentation broth extracted

with 10 mL 80:20 EtoAc/methanol O

Erythromycins from Streptomyces sp. Compounds: 1. Erythromycin A 2. Erythromycin B

99 1 3. Erythromycin A enol ether

1: TOF MS ES+ 1.09e5 %

0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 Min

Analysis of Estradiols using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® H-Class with ACQUITY® PDA

® ® Column: CORTECS UPLC C18, 1.6 µm, 2.1 x 100 mm (p/n 186007095) Separation mode: Isocratic OH OH Mobile phase: 45:55 water/acetonitrile with 0.1% formic acid CH Flow rate: 0.61 mL/min HO HO Detection: UV at 220 nm Estradiol Ethinyl estradiol Injection volume: 1.4 µL

Column temp.: 30 °C O OH CH Standards: Prepared to 20 µg/mL in 50% acetonitrile in water HH Sample: 1 birth control tablet dissolved into HO H H 1 mL of water, filtered through 0.2 µm filter O Estrone Levonorgestrel

Estradiols Compounds: 1. Estradiol 2. Ethinyl estradiol 0.10 1 2 3 3. Estrone 0.08 4. Levonorgestrel

0.06 4 AU Standards 0.04

0.02

0.00

0.20 4

0.15 AU 0.10 Tablet Sample 2

0.05

0.00

0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Min

Analysis of Phenolics in Echinacea using CORTECS UPLC 1.6 µm Columns

CONDITIONS OH ® ® System: ACQUITY UPLC I-Class with ACQUITY PDA HO OH

® ® HO Column: CORTECS UPLC C18, 1.6 µm, 2.1 x 50 mm (p/n 186007093) O O O O OH Mobile phase A: 0.1% trifluoroacetic acid in water O HO O O OH OH Mobile phase B: Acetonitrile HO O O O O HO HO Detection: UV at 330 nm HO OH HO OH O OH OH Flow rate: 0.5 mL/min Caftaric Acid Echinacoside Gradient: See table O HO O OH OH Injection volume: 1.0 µL HO O O HO O Column temp.: 30 °C OH O OH HO O O OH HO O OH Sample diluent: Water OH Chlorogenic acid Cichoric acid

Time OH (min) %A %B Curve O OH Initial 92 8 – HO O O HO 0.10 92 8 6 HO OH O 4.45 50 50 7 HO Cynarin O 4.86 10 90 6 5.00 92 8 6 6.00 92 8 6

Phenolics in Echinacea Compounds: 1. Caftaric acid (100 µg/mL) 2. Chlorogenic Acid (100 µg/mL) 3. Cynarin (100 µg/mL) 4. Echinacoside (100 µg/mL) 5. Cichoric acid (100 µg/mL)

0.60 3

4 0.40 2

AU 1

0.20

0.00

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Min

Analysis of Statins using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® I-Class with ACQUITY® PDA

® ® Column: CORTECS UPLC C18, 1.6 µm, 2.1 x 50 mm (p/n 186007093) Mobile phase A: 0.1% formic acid in water HO O Mobile phase B: 0.1% formic acid in acetonitrile OH HO O

HO O Detection: UV at 248 nm O O H H O O Gradient: See table H H H Flow rate: 0.6 mL/min HO Injection volume: 2.0 µL Pravastatin Lovastatin Column temp.: 30 °C

Sample diluent: 50:50 methanol/water HO O O O H O Time Flow Rate H H (min) (mL/min) %A %B Initial 0.6 95 5 Simvastatin 5.0 0.6 5 95 5.1 0.6 95 5 6.0 0.6 95 5

Statins Compounds: 1. Pravastatin (0.1 mg/mL) 2. Lovastatin (0.1 mg/mL) 1 3. Simvastatin (0.1 mg/mL)

0.80

0.60 AU 0.40

3 0.20

0.00

1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 Min

©2014 Waters Corporation. Waters, The Science of What’s Possible, CORTECS, ACQUITY UPLC, ACQUITY, and UPLC are registered trademarks of Waters Corporation. WA64683 31 [ C18 APPLICATION HIGHLIGHT ] Analysis of Synthetic Cannabinoids from Urine using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® I-Class with Xevo® TQD Mass Spectrometer

® ® Column: CORTECS UPLC C18, 1.6 μm, 2.1 x 100 mm (p/n 186007095) Column temp.: 30 °C Injection volume: 5 μL Flow rate: 0.6 mL/min. Mobile phase A: 0.1% formic acid in Milli-Q® water Mobile phase B: 0.1% formic acid in acetonitrile Vials/plates: 96-well collection plates with 700 µL deactivated glass inserts (p/n 186000349DV)

Time (min) %A %B Curve Initial 70 30 – 2.0 50 50 6 3.0 50 50 6 7.0 10 90 6

7.2 70 30 6 To see the full application note, visit www.waters.com 8.5 70 30 6 and search for literature code: 720004780EN

Calibration Standard at 20 ng/mL Peaks 5–10 by C18

Compounds: 1) AM 2223 7) JWH-018 5-COOH met. 13) JWH-073 19) UR-144 2) RCS4, M10 8) JWH-073 (+/-) 3-OH met. 14) JWH-022 20) JWH-210 3) RCS-4, M11 9) JWH-018 5-OH met. 15) XLR-11 21) AB 001 4) AM 1248 10) JWH-018 (+/-) 4-OH met. 16) JWH-203 22) AKB 48 5) JWH-073 4-COOH met. 11) JWH-015 17) JWH-018 6) JWH-073 4-OH met. 12) RCS-4 18) RCS-8

10 0 100 3 8 1 6 8 6 4 2 9, 10 9, 10 ACQUITY UPLC BEH C18 2.1 x 100 mm

% 5 % 7 5 7

0 0.51.0 1.52.0 2.53.0 3.5 Min. 2.4 2.6 2.8 3.0 3.23.4 Min.

10 0 22 100 6 17 18 8 11, 13, 12 14 19 21 9, 10 CORTECS UPLC C18 2.1 x 100 mm 20 % % 15 5 16 7

0 0 4.04.5 5.05.5 6.06.5 7.0 Min. 2.2 2.4 2.6 2.8 3.03.2 Min.

©2014 Waters Corporation. Waters, The Science of What’s Possible, CORTECS, Xevo, ACQUITY UPLC, and UPLC are registered trademarks of Waters Corporation All other trademarks are property of their respective owners.. WA64699 32 [ C18 APPLICATION HIGHLIGHT ] Analysis of Synthetic Cannabinoids from Whole Blood using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® and ACQUITY® TQD Mass Spectrometer

® ® Column: CORTECS UPLC C18, 1.6 μm, 2.1 x 100 mm (p/n 186007095) Column temp.: 30 °C Injection volume: 10 μL Flow rate: 0.6 mL/min Mobile phase A: 0.1% formic acid in Milli-Q® water Mobile phase B: 0.1% formic acid in acetonitrile Vials/plates: Ostro™ 96-well sample collection plates, 2.0 mL (p/n 186005518)

Time (min) %A %B Curve Initial 70 30 – 2.0 50 50 6 3.0 50 50 6 7.0 10 90 6

7.2 70 30 6 To see the full application note, visit www.waters.com 8.0 70 30 6 and search for literature code: 720004726EN

Calibration Standard at 20 ng/mL

Compounds: 1. AM 2223 6 8 2. RCS4, M10 3. RCS-4, M11 % 9, 10 4. AM 1248 5. JWH-073 4-butanoic acid met 1 5 6. JWH-073 4-hydroxybutyl met 3 4 2 7 7. JWH-018 5-pentanoic acid met 8. JWH-073 (+/-) 3-hydroxybutyl met 0 9. JWH-018 5-hydroxypentyl met 0.5 1.0 1.5 2.0 2.5 3.0 3.54.0 Min. 10. JWH-018 (+/-) 4-hydroxypentyl met 11. JWH-015 12. RCS-4 13. JWH-073 11, 13, 18 14. JWH-022 12 14 15. XLR-11

21 16. JWH-203 17 20 17. JWH-018 % 22 18. RCS-8 19. UR-144 19 15 16 20. JWH-210 21. AB 001 22. AKB 48 0 4.0 4.5 5.0 5.5 6.0 6.5 7.07.5 Min.

©2014 Waters Corporation. Waters, The Science of What’s Possible, CORTECS, ACQUITY UPLC, ACQUITY, and UPLC are registered trademarks of Waters Corporation. Ostro is a trademark of Waters Corporation. All other trademarks are property of their respective owners. WA64700 33 [ C18 APPLICATION HIGHLIGHT ]

Analysis of Abacavir Related Compounds using CORTECS 2.7 µm Columns

CONDITIONS

System: Alliance® HPLC with 2998 Photodiode Array Detector NH2 N N Column: Fully-Porous C18, 5 µm, 4.6 x 150 mm N N NH2 ® CORTECS C18, 2.7 µm, 4.6 x 75 mm (p/n 186007376) HO Mobile phase A: 0.1% trifluoroacetic acid in water 1. Descycleopropylabacavir Mobile phase B: 85% methanol in water

Detection: UV at 254 nm HN HN HN N N N N N N Gradient: See table N N N N NH2 N NH2 N NH2 Column temp.: 30 ˚C HO HO O Sample: Abacavir USP Related Compounds 2. Abacavir 3. 1R,4R Trans abacavir Sample diluent: Water 5. O-t-Butyl-abacavir

Gradient Time (min) HN N 4.6 x 150 mm 4.6 x 75 mm 2.1 x 50 mm %A %B Curve N N Initial Initial Initial 95 5 – N NH2

23.64 6.38 2.52 70 30 6 H2N N O 38.39 10.37 4.09 10 90 11 N NH2 43.83 11.83 4.68 10 90 11 Cl 44.89 12.12 4.69 95 5 11 4. O-(4-Chloro-2,5- diaminopyrimidnyl)-abacavir

Abacavir Related Compounds

Fully-porous C18, 5 µm, 4.6 x 150 mm Acetonitrile Used Flow rate: 1.00 mL/min per Analysis 2 0.020 System: Alliance HPLC 20.6 mL 3 AU 0.010 1 Compounds: S 4 5 1. Descycleopropylabacavir 0.000 2. Abacavir 3. 1R,4R Trans abacavir 12 14 16 18 20 22 24 26 28 30 Min. 4. O-(4-Chloro-2,5-diaminopyrimidnyl)-abacavir 5. O-t-Butyl-abacavir CORTECS C , 2.7 µm, 4.6 x 75 mm s. Solvent peak 18 Flow rate: 1.85 mL/min 2 System: Alliance HPLC 0.020 10.5 mL

AU 0.010 1 3 4 5 S 0.000

3.54.0 4.55.0 5.56.0 6.57.0 7.58.0 Min.

©2014 Waters Corporation. Waters, The Science of What’s Possible, CORTECS, ACQUITY UPLC, ACQUITY, UPLC, and Alliance are registered trademarks of Waters Corporation. WA64697 34 [ C18 APPLICATION HIGHLIGHT ]

Fast Analysis of Antibacterials using CORTECS 2.7 µm Columns

CONDITIONS System: ACQUITY UPLC® H-Class System with ACQUITY® PDA Detector

® Column: CORTECS C18, 2.7 µm, 4.6 x 50 mm (p/n 186007375)

Fully Porous C18, 5 µm, 4.6 x 100 mm O Mobile phase A: 0.1% formic acid in water H N N N N O O Mobile phase B: 0.1% formic acid in acetonitrile N O COOH O Detection: UV at 254 nm O Carbadox Oxolinic acid Gradient: Linear from 5–50% B Column temp.: 30 ˚C NH2 H N N Sample diluent: 20% acetonitrile in water N N S O O COOH N O

Sulfaquinoxaline Nalidixic acid

Antibacterials

Compounds: 2 0.80 Fully-porous C18, 5 µm, 4.6 x 100 mm 1. Carbadox (0.1 mg/mL) Flow rate: 1 mL/min 0.60 2. Oxolinic acid (0.1 mg/mL) 4 Injection volume: 3 µL

AU 0.40 3. Sulfaquinoxaline (0.1 mg/mL) Peak capacity: 60 4. Nalidixic acid (0.1 mg/mL) 1 3 0.20

0.00

0 2 4 6 8 10 12 Min. 0.80

0.60 CORTECS C18, 2.7 µm, 4.6 x 50 mm Flow rate: 1 mL/min Higher Throughput 0.40 AU Injection volume: 1.5 µL Peak capacity: 55 0.20

0.00

024 6 810 12 Min. 0.80 CORTECS C , 2.7 µm , 4.6 x 50 mm 0.60 18 Flow rate: 2 mL/min 0.40 Injection volume: 1.5 µL Greater Speed AU Peak capacity: 55 0.20

0.00

0 2 3 4 6 8 10 12 Min.

©2014 Waters Corporation. Waters, The Science of What’s Possible, CORTECS, ACQUITY UPLC, and ACQUITY are registered trademarks of Waters Corporation. WA64696 35 [ C18 APPLICATION HIGHLIGHT ] Analysis of LCMS Quality Control Reference Material (QCRM) using CORTECS 2.7 µm Columns

CONDITIONS H2N ® HO O System: ACQUITY UPLC with TQD Mass Spectrometer O NH O OH S N ® N NH2 Column: CORTECS C18, 2.7 µm, 4.6 x 100 mm (p/n 186007377) O H H NH2

Mobile phase A: 0.1% formic acid in water Sulfaguanidine Acetaminophen L-leucine Mobile phase B: 0.1% formic acid in acetonitrile OCH3

OO N Flow rate: 2.0 mL/min N S N N OCH3 OH Gradient: See table H Terfenadine OH H2N Injection volume: 9.6 µL Sulfadimethoxine Column temp.: 40 ˚C CN OH H3 CO N OCH 3

Sample: LCMS QCRM (p/n 186006963) O O H CO Verapamil OCH H 3 3 N N OH Capillary voltage: 3.8 kV H O NH2 O O Cone voltage: 30 V N H O Val-Tyr-Val O O N H O Extractor: 3 V H H O O RF lens: 0.0 V Reserpine O Ion energy: 0.5 Time Flow Rate Source temp: 120 °C (min) (mL/min) %A %B Curve Desolvation temp: 450 °C 0.00 2.0 95 5 – 9.60 2.0 25 75 6 Desolvation flow: 1000 L/hr 10.0 2.0 25 75 6 Cone flow: 30 L/hr 10.1 2.0 75 25 6 12.0 2.0 75 25 6

LCMS QCRM Compounds: 100 1. Sulfaguanidine 2. Acetaminophen 3. Val-Tyr-Val 4. Leucine 5. Sulfadimethoxine 3 6. Verapamil 7. Reserpine 8. Terfenadine

% 1 2

6 7 8 4 5

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 Min.

Analysis of Sudan Dyes using CORTECS 2.7 µm Columns

CONDITIONS System: ACQUITY UPLC® H-Class with Capillary voltage: 1.5 kV ® Xevo TQD Mass Spectrometer Desolvation temp.: 500 °C ® Column: CORTECS C18, 2.7 µm, Desolvation gas flow: 1000 L/Hr 2.1 x 100 mm (p/n 186007367) Source temp.: 150 °C Mobile phase A: Water + 0.1% formic acid Mobile phase B: Methanol + 0.1% formic acid Time Flow Rate (min) (mL/min) %A %B %C Curve Mobile phase C: Acetonitrile + 0.1% formic acid Initial 0.4 80 10 10 – Flow rate: 0.4 mL/min 0.5 0.4 40 30 30 6 Gradient: See table 5.0 0.4 0 50 50 6 9.0 0.4 0 50 50 6 Injection volume: 5 µL 9.1 0.4 80 10 10 6 Column temp.: 45 ˚C 12.0 0.4 80 10 10 6 Sample: Sudan Dyes in Oleoresin Sample preparation Oleoresin prepared using Certified Sep-Pak® Silica 3 cc Vac Cartridge, 500 mg sorbent per cartridge, 55–105 µm particle Size) (p/n 186004615) Ionization mode: ESI+

100ppb Spiked Sudan Dyes in Oleoresin

oleor0319_18 MRM of 22 Channels ES+ oleor0319_18 MRM of 22 Channels ES+ 2.94 5.43 100 215.07 > 121.96 (Sudan Orange G ) 100 457.1 > 211.1 (Sudan Black B ) 1.28e5 7.18e5 % % Sudan Orange G Sudan Black B 0 0 2.5 5.0 7.5 10.0 2.5 5.0 7.5 10.0

oleor0319_18 MRM of 22 Channels ES+ oleor0319_18 MRM of 22 Channels ES+ 5.23 5.73 100 279.14 > 123.05 (Sudan Red G ) 100 381.14 > 105.88 (Sudan IV) 5.24 1.39e7 5.04e5 5.28 % % 5.30 Sudan Red G 6.58 Sudan IV 4.14 4.45 7.62 0 0 2.5 5.0 7.5 10.0 2.5 5.0 7.5 10.0

oleor0319_18 MRM of 22 Channels ES+ oleor0319_18 MRM of 22 Channels ES+ 5.49 100 277.1 > 156.04 (Sudan II ) 100 380.14 > 183.08 (Sudan Red 7B ) 4.72e5 2.91e6 % % Sudan Red 7 B Sudan II 5.73 0 0 2.5 5.0 7.5 10.0 2.5 5.0 7.5 10.0

oleor0319_18 MRM of 22 Channels ES+ oleor0319_18 MRM of 22 Channels ES+ 4.57 5.91 100 249.07 > 156.02 (Sudan I ) 100 353.07 > 77.05 (Sudan III ) 2.07e6 1.33e5 % % Sudan I 5.96 Sudan III 2.62 4.05 5.30 0 0 2.5 5.0 7.5 10.0 2.5 5.0 7.5 10.0

oleor0319_18 MRM of 22 Channels ES+ oleor0319_18 MRM of 22 Channels ES+ 3.99 4.12 100 226.14 > 120.06 (Dimethyl Yellow ) 100 294.07 > 143.02 (Para Red ) 1.48e6 5.86e4 % % Dimethyl yellow 1.54 3.78 Para Red 2.94 3.08 4.89 5.94 6.10 0 0 2.5 5.0 7.5 10.0 Min. 2.5 5.0 7.5 10.0 Min.

Analysis of Synthetic Cannabinoids using CORTECS 2.7 µm Columns

CONDITIONS

® System: ACQUITY UPLC I-Class System (SM-FL), (1-(4-hydroxypentyl)-1 H-indol-3-yl) OH (naphthalen-1-yl)methanone Column Manager (CMA) with O N Xevo® TQD Mass Spectrometer

® Column: CORTECS C18, 2.7 µm, O 2.1 x 100 mm (p/n 186007367) N Mobile phase A: 0.1% formic acid in water AB 001 O N OH Mobile phase B: 0.1% formic acid in acetonitrile (1-(5-hydroxypentyl)-1H-indol-3-yl) Gradient: See table (4-hydroxyphenyl)methanone HO Injection volume: 5 µL Column temp.: 30 ˚C Time (min) %A %B Curve Sample: Synthetic Cannabinoid Mix Initial 70 30 – Challenge Sample: Prepared plasma using 3.0 50 50 6 Ostro™ Sample Preparation 4.5 50 50 6 Plate (p/n 186005518) 10.5 10 90 6 13.0 10 90 6 13.1 70 30 11 15.0 70 30 11

Synthetic Cannabinoids Retention Time Peak CompoundInitial InjectionInjection 1000 2RCS-4, M10 1.91 1.90 10 JWH-018, 4-OH met. 4.18 4.18 21 AB 001 10.49 10.48 Pressure 3753 3749

Cann_50_140314_005 100 7: MRM of 2 Channels ES+ 100 4 1 3 9,10 9,10 8 TIC (AM2233) 3 8 6 4.99e6 6 4

2 2 1 % %

5 5 7 7

0 0 123456 Min. 123456 Min.

11, 100 11, 13, 18 100 13, 18 12 14 12 14 22 17 22

20 17 % % 21 20 16 19 21 16 19 15 15

0 0 78910 11 Min. 7891011 Min.

©2014 Waters Corporation. Waters, The Science of What’s Possible, CORTECS, Xevo, ACQUITY UPLC, and ACQUITY are registered trademarks of Waters Corporation. Ostro is a trademark of Waters Corporation. WA64698 38 [ HILIC APPLICATION HIGHLIGHT ] Quantification of Acetylcholine, Histamine, and their Metabolites in Human Cerebrospinal Fluid (CSF) using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® with Xevo® TQ-S Mass Spectrometer Column: CORTECS® UPLC® HILIC, 1.6 µm, O + 2.1 x 100 mm (p/n 186007106) N+ N O HO

Mobile phase A: 100 mM ammonium formate, pH 3 Acetylcholine (ACh) Choline (Ch) Mobile phase B: Acetonitrile NH2 OH N N N Flow rate: 0.5 mL/min O N N HN NH2 Column temp.: 45 °C CH3 CH3 Histamine (HA) tele-Methylhistamine (t-mHA) tele-Methyimidazoleacetic acid (t-MIAA) Sample temp.: 6 °C Injection volume: 10 µL

® ® Collection plates: Waters ACQUITY Time Flow Rate 1-mL collection plates (min) (mL/min) %A %B Curve Initial 10 90 – – 0.75 40 60 6 6 1.00 40 60 6 6 To see the full application note, visit www.waters.com 1.25 70 30 6 6 and search for literature code: 720004722EN 1.90 10 90 11 6

Acetylcholine, Histamine, and their Metabolites t-mHA 1.73 MRM of 10 channels ES+ 126.1 > 109.2 (t-MHA) 3.43e4

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.42.6 2.83.0 3.23.4 Min.

MRM of 10 channels ES HA 112.2 > 95.12 (HA) 1.61 5.39e4

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.42.6 2.83.0 3.23.4 Min.

MRM of 10 channels ES 141.1 > 95.17 (MIAA) t- MIAA 1.14e5

%% % 1.53

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.42.6 2.83.0 3.23.4 Min. Ch 1.45 MRM of 10 channels ES+ 104.2 > 60.1 (CH) 1.30e5

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.42.6 2.83.0 3.23.4 Min.

1.36 MRM of 10 channels ES+ ACh 146.1 > 87.05 (ACH) 6.69e4 %

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.42.6 2.83.0 3.23.4 Min.

©2014 Waters Corporation. Waters, The Science of What’s Possible, CORTECS, Xevo, ACQUITY UPLC, ACQUITY , Xevo, and UPLC are registered trademarks of Waters Corporation. WA64702 39 [ HILIC APPLICATION HIGHLIGHT ]

Analysis of Acyclovir and Guanine using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® with ACQUITY® PDA Column: CORTECS® UPLC® HILIC, 1.6 µm, 2.1 x 100 mm (p/n 186007106) Separation mode: Isocratic (15:85 mobile phase A:B) O Mobile phase A: 0.2% formic acid in water N N OH Mobile phase B: 0.2% formic acid in acetonitrile H2N N N O

Detection: UV at 254 nm Acyclovir Flow rate: 0.6 mL/min

Injection volume: 0.4 µL O HN N Column temp.: 30 °C H N N N 2 H Sample diluent: 0.02 N sodium hydroxide in Guanine 60:40 acetonitrile/water

Acyclovir and Guanine Compounds: 1. Acyclovir (50 µg/mL) 2. Guanine (50 µg/mL)

1 0.08

0.06

AU 0.04

0.02

0.00

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 Min

©2014 Waters Corporation. Waters, The Science of What’s Possible, CORTECS, ACQUITY UPLC, ACQUITY, and UPLC are registered trademarks of Waters Corporation. WA64666 40 [ HILIC APPLICATION HIGHLIGHT ] Analysis of Cold Medicine Compounds by HILIC using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® H-Class with ACQUITY® PDA Column: CORTECS® UPLC® HILIC, 1.6 µm, 2.1 x 100 mm (p/n 186007106) Mobile phase A: 50:50 acetonitrile/10 mM ammonium formate with 0.125% formic acid Mobile phase B: 90:10 acetonitrile/10 mM ammonium formate with 0.125% formic acid Detection: UV at 265 nm H3CO Flow rate: 0.6 mL/min HO O H Gradient: See table N H NCH3 Injection volume: 1.0 µL Acetaminophen Dextromethorphan Column temp.: 30 °C Sample diluent: 75:25 acetonitrile/methanol with 0.2% formic acid Cl N N O

N Time Flow Rate (min) (mL/min) %A %B Chlorpheniramine Diphenhydramine Initial 0.6 0.1 99.9 OH 1.0 0.6 0.1 99.9 H N 2.6 0.6 99.9 0.1 2.7 0.6 0.1 99.9 Pseudoephedrine 3.5 0.6 0.1 99.9

Cold Medicine Compounds Compounds: 1. Acetaminophen (10 µg/mL) 2. Diphenhydramine (100 µg/mL) 1 0.070 3. Dextromethorphan (100 µg/mL) 4. Pseudoephedrine (200 µg/mL) 5. Chlorpheniramine (100 µg/mL) 0.060

5 0.050

0.040 AU

0.030

0.020 3

0.010 2 VO = 0.34 4

0.000

0.0 0.5 1.0 1.5 2.0 2.5 Min

Analysis of Histidine Dipeptides using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® H-Class with Xevo® TQD Mass Spectrometer Column: CORTECS® UPLC® HILIC, 1.6 µm, 2.1 x 50 mm (p/n 186007104) Mobile phase A: 50:50 acetonitrile/10 mM ammonium formate with 0.125% formic acid Mobile phase B: 95:5 acetonitrile/10 mM ammonium formate with 0.125% formic acid Detection: ESI+, scan m/z 100–300 O Flow rate: 0.5 mL/min H H N NH N N NH2 HO O Gradient: See table NH2 N HO O Capillary voltage: 1 kV Creatine Carnosine Cone voltage: 30 V O COOH N N Desolvation gas: 800 L/hr H2N N H Desolvation temp.: 300 °C Anserine Injection volume: 5.0 µL

Sample diluent: 75:25 acetonitrile/methanol with Time Flow Rate 0.2% formic acid (min) (mL/min) %A %B Initial 0.5 0.1 99.9 5 0.5 99.9 0.1 5.01 0.5 0.1 99.9 6 0.5 0.1 99.9

Histidine Dipeptides Compounds: 1 1. Creatine, m/z 132 (1 µg/mL) 2. Carnosine, m/z 227 (1 µg/mL) 3. Anserine, m/z 241 (5 µg/mL) %

2 3

0.0

0.0 1.0 2.0 3.0 4.0 5.0 Min

Analysis of Local Anesthetics by HILIC using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® I-Class with ACQUITY® PDA Column: CORTECS® UPLC® HILIC, 1.6 µm, 2.1 x 50 mm (p/n 186007104) Mobile phase A: 50:50 acetonitrile/10 mM ammonium formate with 0.125% formic acid Mobile phase B: 90:10 acetonitrile/10 mM ammonium formate with 0.125% formic acid Detection: UV at 245 nm Gradient: See table O H N Flow rate: 0.8 mL/min N O N O Injection volume: 5.0 µL H2N Lidocaine Butacaine Column temp.: 30 °C Sample diluent: 75:25 acetonitrile/methanol O O with 0.2% formic acid N O N O N H H2N Time Flow Rate Tetracaine Procaine (min) (mL/min) %A %B

Initial 0.8 0.1 99.9 O N 1.0 0.8 0.1 99.9 N H 2.6 0.8 99.9 0.1 H2N 2.7 0.8 0.1 99.9 Procainamide 3.5 0.8 0.1 99.9

Local Anesthetics Compounds: 1. Lidocaine (100 µg/mL) 2. Butacaine (100 µg/mL) 3. Tetracaine (100 µg/mL) 4. Procaine (100 µg/mL) 1 5. Procainamide (100 µg/mL) 0.20 5 2 4 0.15

0.10 AU

0.05

0.00

-0.05

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Min

Analysis of Morphine Metabolites using CORTECS UPLC 1.6 µm Columns

Flow rate: 1.235 mL/min HO HO Gradient: See table O H

NCH3 O Injection volume: 5 µL H + O N O– Column temp.: 30 °C O HO Sample diluent: Mobile phase B 6-Acetylmorphine Morphine-N-oxide

HO O HO Time Flow Rate OH O (min) (mL/min) %A %B O O H HO HO O H 0.00 1.235 0.1 99.9 NCH3 NCH3 0.51 1.235 0.1 99.9 HO HO

2.11 1.235 99.9 0.1 Morphine Morphine-3β-glucuronide 2.19 1.235 0.1 99.9 3.00 1.235 0.1 99.9

Morphine Metabolites Compounds: 1. 6-Acetylmorphine (0.2 mg/mL) 2. Morphine (0.2 mg/mL) 3. Morphine-N-oxide (0.1 mg/mL) 4. Morphine-3β-glucuronide (0.2 mg/mL)

1 0.20 4

2 0.15

0.10 AU 3

0.05

0.00

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Min

Analysis of Nucleic Acid Bases using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® I-Class with ACQUITY® PDA Column: CORTECS® UPLC® HILIC, 1.6 µm, 2.1 x 50 mm (p/n 186007104) Mobile phase A: 50:40:10 acetonitrile/methanol/20 mM ammonium acetate with 0.05% acetic acid Mobile phase B: 90:5:5 acetonitrile/methanol/4 mM ammonium acetate with 0.01% acetic acid Detection: UV at 254 nm O NH2 S Flow rate: 0.45 mL/min HN N H N NH Gradient: See table 2 2 O N O N H H Injection volume: 0.4 µL Thiourea Thymine Cytosine Column temp.: 30 °C

Sample diluent: 75:25 acetonitrile/methanol with 0.2% formic acid O NH2 F HN N

O N O N HO H Time Flow Rate O (min) (mL/min) %A %B 5-Fluorocytosine OH 0.00 0.45 0.1 99.9 Thymidine 0.51 0.45 0.1 99.9 2.11 0.45 99.9 0.1 2.19 0.45 0.1 99.9 3.00 0.45 0.1 99.9

Nucleic Acid Bases Compounds: 1. Thiourea (0.05 mg/mL) 2. Thymidine (0.1 mg/mL) 3. Thymine (0.1 mg/mL) 1 4. 5-Fluorocytosine (0.1 mg/mL)

0.40 5. Cytosine (0.1 mg/mL) 5

0.30 4 2

AU 0.20

0.10

0.00

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 Min

©2014 Waters Corporation. Waters, The Science of What’s Possible, CORTECS, ACQUITY UPLC, ACQUITY, and UPLC are registered trademarks of Waters Corporation. WA64663 45 [ HILIC APPLICATION HIGHLIGHT ] Analysis of Paraquat and Diquat in Drinking Water using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® H-Class with photodiode array (PDA) detection with ACQUITY® TQD Mass Spectrometer Column: CORTECS® UPLC® HILIC, 1.6 µm, 2.1 x 100 mm (p/n 186007106) Separation mode: Isocratic (50:50 mobile phase A:B) + + Mobile phase A: 200 mM ammonium formate buffer at pH 3.7 CH3 NCN H3

Mobile phase B: Acetonitrile Paraquat Injection volume: 20 µL Column temp.: 30 °C

Flow rate: 0.5 mL/min N+ N PDA detection: Diquat UV at 308 nm, paraquat UV at 257 nm + Diquat Sample vials: Polypropylene autosampler vials (p/n 186002642)

MRM Transitions

Compound MRM Cone (V) CID (eV)

Diquat 183.1 > 157.1 50 25 183.1 > 130.1 50 30 Paraquat 185.1 > 170.1 38 22 To see the full application note, visit www.waters.com 171.1 > 77.0 45 40 and search for literature code: 720004732EN

Paraquat and Diquat in Drinking Water

(A) ACQUITY UPLC BEH HILIC Column (B) CORTECS UPLC HILIC Column

95 Diquat 95 Diquat % % Paraquat

Paraquat

-5 -5 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Min. 0.0 0.5 1.0 1.52.0 2.53.0 3.54.0 4.55.0 5.5 Min.

©2014 Waters Corporation. Waters, The Science of What’s Possible, CORTECS, ACQUITY, ACQUITY UPLC, Xevo, and UPLC are registered trademarks of Waters Corporation. WA64703 46 [ HILIC APPLICATION HIGHLIGHT ] Determination of Paraquat and Diquat in Potato and Wheat Using the CORTECS UPLC 1.6 µm Column

CONDITIONS System: ACQUITY UPLC® H-Class with ACQUITY® TQD Mass Spectrometer Column: CORTECS® HILIC, 1.6 µm, 2.1 x 100 mm (p/n 186007106) Isocratic: 50:50 A/B Mobile phase A: 200 mM ammonium formate buffer at pH 3.7 + + Mobile phase B: Acetonitrile CH3 NCN H3

Injection volume: 20 µL Paraquat Column temp.: 30 °C Wash solvent: 50:50 acetonitrile/water Purge solvent: 50:50 acetonitrile/water N+ N + Flow rate: 0.5 mL/min Diquat Sample vials: Polypropylene autosampler vials (p/n 186002642) MRM Transitions Ionization mode: Positive Electrospray Source temp.: 150 °C Compound MRM Cone (V) CID (eV) Desolvation temp.: 350 °C Diquat 183.1 > 157.1 50 25 183.1 > 130.1 50 30 Desolvation gas flow: 800 L/hr Paraquat 185.1 > 170.1 38 22 Cone gas flow: 30 L/hr 171.1 > 77.0 45 40 Collision gas flow: 0.20 mL/min

Paraquat and diquat in potato

Quats_19Aug2013_02 Sm (Mn, 2x2) MRM of 4 Channels ES+ 183.1 > 157.1 (Diquat) 95 2.44e3 %

-5 1.2 1.4 1.6 1.8 2.0 2.22.4

Quats_19Aug2013_02 Sm (Mn, 3x2) MRM of 4 Channels ES+ 185.1 > 170.1 (Paraquat) 95 647 %

-5 1.2 1.4 1.6 1.8 2.0 2.22.4 Min.

Analysis of Water Soluble Vitamins using CORTECS UPLC 1.6 µm Columns

CONDITIONS System: ACQUITY UPLC® with ACQUITY® PDA

Column: CORTECS® UPLC® HILIC, 1.6 µm, 2.1 x 50 mm (p/n 186007104) CH 2 OH HO H Mobile phase A: 50:50 acetonitrile/10 mM ammonium formate with 0.125% formic acid HO H HO H Mobile phase B: 90:10 acetonitrile/10 mM ammonium formate with 0.125% formic acid CH 2 N N O Detection: UV at 265 nm NH N Flow rate: 0.5 mL/min H Folic acid O H2N N N H Riboflavin Gradient: See table N N N H O N COOH Injection volume: 5.0 µL O COOH O CN Column temp.: 30 °C H 2 N O NH 2 O Sample diluent: 75:25 acetonitrile/methanol H 2 N N N N O Co + NH 2 with 0.2% formic acid N H 2 N N NH 2 O O NH H 2 Time Flow Rate Nicotinamide N O O O O (min) (mL/min) %A %B P N + O – N Initial 0.5 0.1 99.9 N O HO OH 5.0 0.5 99.9 0.1 O 5.1 0.5 0.1 99.9 O HO 6.0 0.5 0.1 99.9 Nicotinic acid Vitamin B12

Water Soluble Vitamins Compounds: 1. Nicotinamide (25 µg/mL) 2. Folic acid (50 µg/mL) 0.80 1 3. Nicotinic acid (50 µg/mL) 4. Riboflavin (25 µg/mL) 0.70 5. B12 (50 µg/mL)

0.60 3

0.50

AU 0.40

0.30

0.20 4

0.10 2 5

0.00

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 Min

Analysis of Basic Drugs in River Water using CORTECS 2.7 µm Columns

CONDITIONS System: ACQUITY UPLC® H-Class with OH Xevo® TQD Mass Spectrometer H H N NH N N 2 HO ® NH NH Column: CORTECS HILIC, 2.7 µm, HO 2.1 x 100 mm (p/n 186007382) Metformin Salbutamol Flow rate: 0.28 mL/min

OH Mobile phase A: Water + 0.1% acetic acid/ammonium N H H Cl N N N acetate buffer (1 g/L water) S N N N H2N H H Mobile phase B: Acetonitrile Cl Cimetidine Clenbuterol Gradient: See table

Column temp.: 45 ˚C H H O N N N S Sample: Basic drugs from river water NO2 Sample preparation 200 mL river water Ranitidine (pH 5.0 with acetic acid) prepared using Oasis® MCX 6 cc Vac Cartridge, 150 mg sorbent per cartridge, 30 µm particle size) 25 ppt Spiked River Water (p/n 186000256 ) CorSil0326_17b MRM of 10 Channels ES+ 6.91 100 315.16 > 176.072 (Ranitidine) Ionization mode: ESI+ 1.82e5 % Ranitidine Capillary voltage: 1.5 kV 0 2.5 5.0 7.5 10.0 12.5 Desolvation temp.: 500 °C CorSil0326_17b MRM of 10 Channels ES+ 5.49 100 277.266 > 202.949 (Clenbuterol) Desolvation gas flow: 1000 L/Hr 1.61e5

% Clenbuterol Source temp.: 150 °C 0.31 0 2.5 5.0 7.5 10.0 12.5

CorSil0326_17b MRM of 10 Channels ES+ 3.93 Time Flow Rate 100 253.074 > 95.013 (Cimitidine) (min) (mL/min) %A %B Curve 1.05e6 % Cimetidine Initial 0.280 2 98 – 0 2.5 5.0 7.5 10.0 12.5 4.50 0.280 30 70 6

CorSil0326_17b MRM of 10 Channels ES+ 10.80 0.280 30 70 6 6.03 100 240.074 > 148.038 (Salbutamol) 3.10e5 11.25 0.280 2 98 6 % Salbutamol

14.40 0.280 2 98 6 0 2.5 5.0 7.5 10.0 12.5 4.50 0.280 2 98 6 CorSil0326_17b MRM of 10 Channels ES+ 7.00 100 130.074 > 60.013 (Metformin) 2.25e5 7.04

% 4.23 Metformin 7.10 4.40 6.15 9.98 12.01 0 2.5 5.0 7.5 10.0 12.5 Min.

©2014 Waters Corporation. Waters, The Science of What’s Possible, ACQUITY UPLC, Xevo, Oasis, and CORTECS are registered trademarks of Waters Corporation. WA60707 49 [ HILIC APPLICATION HIGHLIGHT ] Analysis of HILIC Quality Control Reference Material (QCRM) using CORTECS 2.7 µm Columns

CONDITIONS

® System: ACQUITY UPLC with PDA Detector O Column: CORTECS® HILIC, 2.7 µm, HN O N 4.6 x 100 mm (p/n 186007392) H Mobile phase: 90:10 acetonitrile/100 mM ammonium Acenaphthene Thymine formate (pH 3.0) (v/v)

NH2 Flow rate: 2.0 mL/min NH2 HN N N Separation: Isocratic N O N N H Injection volume: 9.6 µL Adenine Cytosine Column temp.: 30 ˚C Sample: HILIC QCRM (p/n 186007226)

HILIC QCRM Compounds: 1. Acenaphthene (19 µg/mL) 2. Thymine (3.7 µg/mL) 3. Adenine (3.7 µg/mL) 0.08 4. Cytosine (7.7 µg/mL)

0.07 3 1 0.06

2 0.05 4

0.04

0.03

0.02

0.01

0.00

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 Min.

Analysis of Morphine Metabolites using CORTECS 2.7 µm Columns

CONDITIONS System: Alliance® HPLC with 2998 Photodiode Array Detector Column: CORTECS® HILIC, 2.7 µm, 4.6 x 150 mm (p/n 186007393) Mobile phase A: 10 mM ammonium formate in 50% acetonitrile/49.875% water/0.125% formic acid Mobile Phase B: 10 mM ammonium formate in 90% acetonitrile/9.875% water/0.125% formic acid Detection: UV at 280 nm

Flow rate: 1.99 mL/min HO HO Gradient: See table O H

NCH3 O Injection volume: 14.4 µL H + O N O– Column temp.: 30 ˚C O HO Sample: Morphine Metabolites 6-Acetylmorphine Morphine-N-oxide Sample diluent: Mobile phase B HO O HO OH O O O H Time HO HO O H NCH (min) %A %B Curve 3 NCH3 Initial 0.1 99.9 – HO HO 4.42 0.1 99.9 6 Morphine Morphine-3β-glucuronide 18.29 99.9 0.1 6 18.99 0.1 99.9 11 26.01 0.1 99.9 11

Morphine Metabolites Compounds: 1. 6-Acetylmorphine (0.2 mg/mL) 2. Morphine (0.2 mg/mL) 3. Morphine-N-oxide (0.1 mg/mL) 4. Morphine-3β-glucuronide (0.2 mg/mL)

2 0.20

0.10

0.08 1 3 0.06 AU

0.04

4 0.02

0.20

0 2 4 064 8.4 10 12 14 Min.

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Waters Corporation Waters, The Science of What’s Possible , CORTECS, ACQUITY UPLC, ACQUITY, UPLC, Xevo, and MassLynx 34 Maple Street are registered trademarks of Waters Corporation. Ostro is a trademark of Waters Corporation. Milford, MA 01757 U.S.A. All other trademarks are the property of their respective owners. T: 508 478 2000 F: 508 872 1990 ©2014 Waters Corporation. Produced in the U.S.A. www.waters.com May 2014 720004739EN IH-PDF