© Advanced Chromatography Technologies Ltd. 1 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Artificial Sweeteners Global Method

1. Acesulfame K 2. Cyclamate 5 3. Saccharin 4. Sucralose 5. Aspartame 6. Neotame 7. Alitame 2 8. Neohesperidin dihydrochalcone 9 9. Dulcin

4 ACE C18, 5μm, 250 x 4.6mm 7 6 Gradient analysis A: Water 8 B: Acetonitrile C: 0.1% TFA Time (mins) %A %B %C 0 88 2 10 25 50 40 10 30 30 60 10 35 88 2 10 3 Flow rate: 1ml/min 1 Column temperature: 30°C Injection volume: 50μl Detection: Corona CAD

Data courtesy of Durham County Council Scientific Services, UK © Advanced Chromatography Technologies Ltd. 2 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Chocolate Analysis

ACE Excel C18-Amide mAU 2mm, 100 x 2.1 mm Gradient analysis

350 5-85%B in 1.5mins

Flow rate: 1.2 mL/min

Temperature: 42°C

300

Detection: 254 nm Ethylparaben

MPA: 10mM HCOONH , pH2.8 (aq)

4 Theophylline Hydroxybenzoic acid Hydroxybenzoic

MPB: 10mM HCOONH4, pH2.8 in 9:1 -

4

Sorbic acid Sorbic Methylparaben

250 v/v MeCN:water

Propylparaben

Caffeine

200 Theobromine

Ethylvanillin

Acesulfam K Acesulfam Vanillin

150 acid Chlorogenic

Epicatechin

Catechin

Guaiacol

100

Saccharin

50 Quercetin

0

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 min

Reproduced with permission of G.J. Schad, Shimadzu Europa GmbH. Analysed on Shimadzu Nexera X2. © Advanced Chromatography Technologies Ltd. 3 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Aminoglycosides in Eggs

UHPLC-MS/MS method for aminoglycosides in eggs Key Extraction at low pH, clean-up with WCX SPE cartridge GNT Gentamicin TBM Tobramycin KNM Kanamycin DHS Dihydrostreptomycin Egg sampleEgg sample spiked spiked at 100 μatg/kg 100 (CC mg/kgα) (CCa) STR Streptomycin HGB Higromycin-B GNT-C1 (478 > 322) RT: 8.40 SPT Spectinomycin

GNT-C2 (464 > 322) RT: 8.31 RT: 8.40 GNT-C2 GNT-C2a ACE Excel C18-PFP, 2μm, 100 x 2.1mm Gradient analysis GNT-C1a (450 > 322) RT: 8.26 A = 20mM HFBA in H2O/CH3CN (98:2) B = 20mM HFBA in CH3CN/H2O (98:2) TBM (468 > 163) RT: 8.09 Time (mins) %B Curve 0 5 - 2 15 6 KNM (485 > 163) RT: 7.52 4.5 19 6 5.5 19.5 8 6 22 6 DHS (584 > 263) RT: 5.65 7 35 6 9 48 8 STR (582 > 263) 9.5 5 6 RT: 5.48 Flow rate: 0.4ml/min Column temperature: 40°C HGB (528 > 177) RT: 5.09 Positive ESI MRM (transitions as shown)

SPT (351 > 333) RT: 3.67

Reproduced with permission of Veterinary Drug Residues Department, Centro Nacional de Alimentacion (CAN, AECOSAN), Madrid, Spain © Advanced Chromatography Technologies Ltd. 4 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Isoflavones in Red Clover and Soy Extract

Extract from red clover and soy dietary supplement Sample preparation mAU 30mg of homogenised capsules/tablets extracted into 3ml

DMSO-H2O (3:1) by ultrasonication. After centrifuging, 1ml of supernatant

was diluted with 4ml DMSO-H2O (3:1)

HPLC Analysis

ACE C18-AR, 3μm, 150 x 2.1mm Gradient analysis

A: H2O – CH3COOH, pH 2.8 B: CH3CN + 0.6% CH3OOH Time (mins) %B 0 15 7 15 27 75 Flow rate: 0.35ml/min Column temperature: 25°C Detection: UV, 254nm Injection volume: 3μl

mins

K. Weinfurter et al. Forsch. Komplementmed. 21 (Suppl.1): 45 (2014) © Advanced Chromatography Technologies Ltd. 5 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Artificial Sweeteners (Stevia Glycosides)

Rebaudioside MW = 967.01

Stevioside MW = 804.87

ACE Excel SuperC18, 2um, 150 x 2.1 mm Gradient analysis

MP A: 10 mM NaH2PO4, pH 2.8 in H2O. MP B: 10 mM NaH2PO4, pH 2.8 in MeCN/H2O (80:20 v/v). 0.6mL/min 39.5 – 48%B in 4 mins. 50C, 1uL. λ = 200 nm. Cycle time = 7 mins.

Reproduced with permission. G.J. Schad and B. Richrath, Shimadzu Europe GmbH. Analysed on Shimadzu Nexera X2. http://images2.advanstar.com/PixelMags/lcgc-eu/pdf/2013-10-sp.pdf © Advanced Chromatography Technologies Ltd. 6 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Mycotoxins / Aflatoxins1 From Peppers

ACE C18-PFP, 3um, 150 x 4.6 mm

Isocratic analysis

Flow rate: 1 mL / min Temperature: 25°C Aflatoxin G1 Detection: Fluorescence Ex λ = 362nm Em λ = 425 nm Mobile phase: 6:4 v/v water:MeOH Injection: 100 uL

Aflatoxin G2

Aflatoxin B1

Reproduced with permission from Public Health Laboratory of Soria, Spain. © Advanced Chromatography Technologies Ltd. 7 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Fingerprinting of Liquorice Extracts

Methanolic liquorice leaf extract at different wavelengths

260nm Sample preparation Plant material ground to fine powder in pestle and mortar. 280nm Powdered material extracted into methanol by ultrasonication for 30 minutes, followed by centrifugal filtration.

330nm HPLC analysis ACE 3 C18-PFP, 3μm, 150 x 2.1mm Gradient analysis 350nm A: Ammonium acetate in water, pH 4 B: Methanol Time (mins) %B 0 10 Comparison of methanolic extracts at 260nm 1 10 11 15 Liquorice leaf 55 90 60 100 Flow rate: 0.4ml/min Column temperature: 40°C Liquorice root Injection volume: 2μl Detection: UV at 260, 280, 330 and 350nm

Reproduced with permission of Unilever, Colworth Science Park, UK © Advanced Chromatography Technologies Ltd. 8 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Sennosides in Traditional Chinese Medicine

Herbal tea bag containing Folium Sennae, Peppermint, Folium Mori, Folium Nelumbinis, Glycyrrhiza Uralensis and Lalang Grass Rhizome

1,000 ACE C18-PFP 3μm, 150 x 4.6mm mAU Gradient analysis

A = 0.75% CH3COOH in H2O 800 Sennoside B B = CH3CN/CH3OH (90:10) Time (mins) %B

600 Sennoside A 0 9 23 28

40 28 400 Flow rate: 0.6ml/min Column temperature: 35°C

Absorbance [mAU] Absorbance Detection: UV, 271nm 200

Sample 2 0

-200 Standard 1

min -400 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 Retention Time [min]

Sennoside A Sennoside B

Reproduced with permission of Vercotech Inc., Taiwan © Advanced Chromatography Technologies Ltd. 9 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Beverage Analysis: Wine Acids

OH O HO • Polar acid resolution OH Malic acid Tartaric acid OH • 100% aqueous eluent O OH OH O Lactic acid HO mAU HO CH3 20 OH O O HO 17.5 HO 1 4 O O 15 Acorbic acid HO OH ACE Excel C18-Amide 12.5 HO O 5 250x2.1mm, 3mm Isocratic analysis 10 O OH 2 3 Oxalic acid 6 40 mM NH4H2PO4, pH 2.5 (aq) Citric acid 7.5 25C O OH OH O 0.21mL/min

5 O OH OH

2.5

0

-2.5

1 2 3 4 5 6 7 8 9 min © Advanced Chromatography Technologies Ltd. 10 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Food & Beverage Additives: Vanillins ACE Excel C18-Amide Vanillin 4-Hydroxy benzaldehyde HO HO 4-Hydroxy benzoic acid H H • Polar acids / aldehydes resolution HO CH3O O 4 O • Enhanced polar retention

CO2H HO Guaiacol

HO HO Eugenol Vanillic acid 1 2 CH O 3 Ethyl vanillin 150x4.6mm, 3mm CH O CO H 3 HO 3 2 CH3O Gradient analysis 6 5 H CH3CH2O A = 0.1% v/ formic acid (aq) O 7 B = 0.1% v/v formic acid in MeCN 40C 1.0mL/min

T (min) %B ACE C18 4 0 30 10 55 10.5 30 15 30 2 1 3 5,6

7

2 4 6 8 10 min © Advanced Chromatography Technologies Ltd. 11 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Separation of Milk Proteins

Intens. 091105.LC4_uTQ.TMG.163_6_01_266.d: Base Peak UV Chromatogram, 214 nm [mAU] MW ~23-25kDa α caseins ACE 5 C18-300 125 MW ~24kDa 5μm, 150 x 2.1mm β caseins Gradient analysis A = 0.01% TFA in water B = 0.01% TFA in acetonitrile Time (mins) %B 100 0 33 5 33 9 35 18 37

75 22 40 MW ~18kDa 27.5 41 β lactoglobulins 28 41 43 43

50 Flow rate: 0.2ml/min Column temperature: 45°C Detection: UV, 214nm

25

0 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 Time [min]

Reproduced with permission of Trevor Gibson, The Chemical Analysis Facility, University of Reading, UK © Advanced Chromatography Technologies Ltd. 12 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Paraben Preservatives

1 ACE 3 Phenyl, 150 x 2.1mm Isocratic

Methanol – 25mM NH4OAc 2 (50:50), pH 6.8 Flow rate: 0.2ml/min Column temperature: 40°C Detection: UV at 240nm 3 Sample volume: 2μl

5 4

1. Methylparaben 6 2. Ethylparaben 3. n-Propylparaben 4. i-Butylparaben 5. n-Butylparaben 6. Benzylparaben

Reproduced with permission of Trevor Gibson, The Chemical Analysis Facility, University of Reading © Advanced Chromatography Technologies Ltd. 13 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Water Soluble Vitamins ACE 3 C18-AR Pantothenic acid (vitamin B5) Cyanocobalamin (vitamin B12) H OH NH OH HO H C 3 CH3 O O D-Biotin (vitamin B7 or vitamin H) O 150x4.6mm, 3mm mAU HN NH 3 p-Aminobenzoic acid Gradient analysis 200 H H O Pyridoxine (vitamin B6) HO O

N CH3 S OH A = 20mM KH2PO4, pH 2.83 (aq) B = 20mM KH2PO4, pH 2.83 in 175 HO OH 1:1 v/v MeOH:water 40C H2N NH2 205nm 1.5mL/min 150 1 HO O O Folic acid (vitamin B9 or vitamin M) O T (min) %B O NH 0 20 125 H3C N N HO NH HN NH 15 70 4 O H3C N N O H2N N N 100 HO Riboflavin (vitamin B2)

6 HO OH OH 7 75 1. Pyridoxine (vitamin B6) 2 5 2. Pantothenic acid (vitamin B5) 3. p-Aminobenzoic acid 50 4. Folic acid (vitamin B9 or vitamin M) 5. D-Biotin (vitamin B7 or vitamin H) 6. Cyanocobalamin (vitamin B12) 7. Riboflavin (vitamin B2) 25

0

0 2 4 6 8 10 12 14 16 min © Advanced Chromatography Technologies Ltd. 14 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Water Soluble Vitamins / Polar Molecules

HO

O O Nicotinamide HO O H Isonicotinamide HO OH O ACE C18-AR, 3um, 150 x 4.6 mm NH H2N O 2 L-Ascorbic acid Isocratic analysis or vitamin C NH N HO N O Flow rate: 1.0 mL/min H mAU O N Temperature: 22°C Orotic acid Detection: UV 260 nm NH2 2 + N N MP: 96.5:3.5 v/v 0.1% H3PO4 (aq) : MeOH S O 10

H3C N H C 7 N 3 NH HO O Thiamine or 3 vitamin B1 N N OH H Hypoxanthine

NH2 4-aminobenzoic acid 4 8 O

HO OH 5 H3C N Pyridoxal or vitamin B6 Pyridoxamine or 1 6 OH OH Vitamin B6 CH3

N CH3 9 HO N HO Pyridoxine or OH vitamin B6

H2N

0 2 4 6 8 min © Advanced Chromatography Technologies Ltd. 15 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Arsenolipids from Edible Seaweed by LC-ICP-MS and LC-ESI-MS

Separation of arsenic species from methanolic extract of the edible seaweed Alaria esculenta

Arsenic-containing hydrocarbon: Arsenic-containing phospholipids:

m/z 389 [M + H]+ for C21H46AsO m/z 959 [M + H]+ for C45H89AsO14P (C16:0/C16:0) m/z 987 [M + H]+ for C47H93AsO14P (C18:0/C16:0) m/z 1015 [M + H]+ for C49H97AsO14P (C20:0/C16:0)

ACE C18, 3μm 150 x 4.6mm Gradient analysis 2.E+07 10000000 A = 0.1% formic acid in H O 389 2 959 B = 0.1% formic acid in CH3OH 9000000 ESI-MS 987 Time (mins) %B 1015 0 0 1.E+07 As 8000000 20 100

7000000 45 100

Flow rate: 1ml/min MS

MS) 5.E+06 6000000 Injection volume: 100μl

- - Split ratio: 75% ESI-MS: 25% ICP-MS

5000000 Thermo Scientific Element 2 ICP-MS

0.E+00 4000000 Mode: Organic mode relative relative intensityICP relative relative intensity(ES Medium resolution ICP-MS 3000000 m/z 75 Thermo Scientific Orbitrap Discovery Positive ESI mode -5.E+06 2000000 Spray voltage: 4.5kV

1000000 Capillary temperature: 320°C Capillary voltage: 42V

-1.E+07 0 0 5 10 15 20 25 30 35 40 45 50 retention time (min)

Reproduced with permission of Dr Andrea Raab, Trace Element Speciation Laboratory, University of Aberdeen, UK © Advanced Chromatography Technologies Ltd. 16 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Water Soluble Vitamins In Green Vegetables By LC-MS/MS

ACE C18, 3μm, 100 x 2.1mm Gradient analysis A: 10mM ammonium acetate (aq), pH 4.5 B: 0.1% acetic acid in methanol C: 0.3% acetic acid in methanol Ascorbic acid Time (mins) %A %B %C 0 90 10 0 Thiamine 3 90 10 0 4 50 0 50 7 50 0 50 Pyridoxine 10 0 100 0 Flow rate: 0.2ml/min Injection volume: 10μl Nicotinamide Column temperature: 20°C TSQ triple quad MS; SRM mode Detection: -ESI for vitamin C Pantothenic acid +ESI for B vitamins

Analyte Transition LOQ Hippuric acid (IS) m/z (ng/mL) Ascorbic acid (Vit C) 174.9  115.2 128.3 Folic acid Thiamine (Vit B1) 265.1  122.1 2.4 Pyridoxine (Vit B6) 169.9  152.1 0.6 Riboflavin Nicotinamide (Vit B3) 123.0  80.3 13.2 Pantothenic acid (Vit B5) 220.0  202.1 23.3

Folic acid (Vit B9) 442.0  294.9 1.9 Riboflavin (Vit B2) 377.1  243.0 0.2

Reproduced with permission of Foodomics Laboratory, CIAL-CSIC, Madrid, Spain Hippuric acid (IS) 180.1  105.2 14.9 17 © Advanced Chromatography Technologies Ltd. ® ACE HPLC / UHPLC Columns www.ace-hplc.com Chloramphenicol in Milk by LC-MS/MS

TIC of 3 MRMs of 0.02 ppb chloramphenicol spiked MRM chromatograms of 0.05 ppb in milk matrix chloramphenicol in milk

m/z 320.9 → 152

m/z 320.9 → 257

m/z 320.9 → 194 ACE C18 3μm, 50 x 2.1mm Gradient analysis A = Water B = Methanol T (mins) %B T (mins) %B 0 10 3.0 95 0.05 10 3.1 95 LOQ Chloramphenicol = 0.02 ppb 2.5 95 4.5 10 Flow rate: 0.5ml/min Injection volume: 10μl Transitions: Bruker EVOQ Elite triple quad MS m/z 320.9 → 152 Quantification VIP heated-ESI temperature: 400°C m/z 320.9 → 257 Qualification Cone gas temperature: 350°C Chloramphenicol m/z 320.9 → 194 Qualification Spray voltage: -4500V

Reproduced with permission of Bruker UK Ltd © Advanced Chromatography Technologies Ltd. 18 ® ACE HPLC / UHPLC Columns www.ace-hplc.com LC-MS/MS of phytoestrogens from hop extracts

Hop extracts obtained by pressurised liquid extraction using ASE 200 system 28e+5 Isoxanthohumol 26e+5 ACE C18-AR, 3μm, 150 x 4.6mm Xanthohumol Gradient analysis

24e+5 A: 1% formic acid in acetonitrile Intensity 22e+5 B: 1% formic acid in methanol C: 1% formic acid in water 20e+5 D: Methanol Time (mins) %A %B %C %D 18e+5 0 56 0 44 0 16e+5 8 51 5 44 0 10 51 5 44 0 14e+5 17 95 5 0 0 12e+5 22 95 0 0 5 Flow rate: 0.6ml/min 10e+5 TSQ-Quantum triple quad ESI 8e+5 Spray voltage: -4500V Precursor ion: 355.4 [M+H]+ 6e+5 MRM transition ions: 179 and 299 4e+5 Collision energy: 28 and 16V

2e+5

0 2 4 6 8 10 12 14 16 LOQ isoxanthohumol : 0.07μg/ml Time (min) LOQ: xanthohumol: 0.01μg/ml

Reproduced with permission of Institute of Food Science Research, University of Madrid, Spain