Food for Thought - Universidad De...

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© 2000 Waters Corp

Food for Thought

Analysis of Pesticides,Nutraceuticals,

Endocrine Disruptors& Related CompoundsUsing HPLC & LC/MS

Waters Corporation

Title Slide

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© 2000 Waters Corp

Agricultural ChemicalsResidues in Food & Water

Fine ChemicalsPesticidesPolymer Additives

Functional Foods & Dietary SupplementsNutrientsNutraceuticals

Environmental ContaminantsEndocrine Disruptors

The environmental Recycle symbol is used here to represent the interrelationshipbetween the topics to be discussed in this seminar.

The Fine Chemicals industry produce pesticides for the AgChem industry andpolymer additives for food packaging. The pesticides are spayed on crops to protectthem but also result in residues found in food and ground water. More freshproduce and other foods are wrapped in plastic packages to maintain freshness andminimize contamination. However, some of the polymer additives used in themanufacturing process are easily extracted into the food.

Nutraceuticals refers to foods or components of foods that may provide healthbenefits. Categories include: Dietary Supplements such as Health foods,Vitamins, Botanicals, and Amino Acids; Functional Foods such as NutritionalBeverages and Dietary Fiber.

The environmental fate of all of these products is that they exist in the air we breath,the water we drink, or the food that we eat. There is a growing concern of the longterm effect these chemicals may have on humans and our endocrine system,especially children.

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© 2000 Waters Corp

Seminar Outline

• Introduction - The apple

• Simple Sugars - HPLC with RI Detection

• Endocrine Disruptors - Sample preparation

• PAH’s - HPLC with PDA & FluorescenceDetection

• Pesticides/Carbamates -Analysis by HPLCwith post column derivatization & API LC/MS

Here is an outline of the topics that will be discussed today.

An apple was used as an example for determining some of the nutrients (simplesugars) in food as well as pesticide/carbamate residues that can be found..

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© 2000 Waters Corp

Seminar Outline (Cont.)

• Polymer Additives - Comparison of LC/MSInterfaces

• Nutraceuticals & Herbal Extracts - Analysisby LC/MS

Seminar outline continued.

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© 2000 Waters Corp

Simple Sugar Analysis by HPLC UsingRefractive Index Detection

Jim Krol

Market Development

Waters Corporation

Introduction to sugar analysis application. This work was performed on a WatersAlliance HPLC System consisting of a 2690 Separations Module, 2410 DifferentialRefractive Index Detector, and Millennium32 chromatography software.

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© 2000 Waters Corp

M inutes2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

Analysis of Mono/DiSaccharidesWaters Carbohydrate Analysis Column

FiveChromatogram

Overlay

1

2

3

Red Delicious Apple9.24g / 100 mL

1 Fructose = 5.16 ± 0.01%2 Glucose = 2.31 ± 0.01%3 Sucrose = 0.09 ± 0.05%

47

µR

I U

nits

Analysis of mono/disaccharide's; fructose, glucose, and sucrose in an apple. Theexcellent reproducibility of the 5 overlay chromatograms demonstrates theexceptional flow characteristics of the of the 2690 Separations Module.

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© 2000 Waters Corp


! Nutritional Labeling of Food Products requireslisting sugar and total carbohydrate content

! Common Sugars defined as• Monosaccharides: Fructose and Glucose• Disaccharides: Sucrose, Maltose, Lactose

! AOAC HPLC Methods Recommend the use ofPropyl Amine functional columns for analysis ofmono and disaccharides in food products

This slide presents the guidelines for sugar determination according to theNutritional Labeling & Education Act passed by the U.S. Congress. Note thatlabeling is not required until the levels are quite high (0.5g total sugar per servingsize) and that only the mono-saccharides fructose, glucose and the di-saccharidessucrose, maltose and lactose need to be labeled. These five sugars are the mostcommon in food systems.

In the Official Methods of Analysis of AOAC International (Association ofOfficial Analytical Chemists), the HPLC Methods recommend the use of ananalytical column containing a silica stationary phase with a propyl aminefunctionality.

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© 2000 Waters Corp

M inutes2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

Fructose

Sucrose

MaltoseLactose

Column : Carbohydrate, 3.9mm x 30 cmMobile Phase: 75% AcCN / 25% WaterFlow Rate: 1.4 mL/min at 35°C; BP = 680 psiDetection: 2410 Refractive Index, 40°C, 128XInjection: 20 µL

Glucose

0.25% Each Sugar

33

µ R

I U

nits


The Waters Carbohydrate Analysis Column incorporates propyl aminefunctionality. Baseline resolution of the mono/disaccharides is obtained using thechromatographic conditions described above. Also available, the Waters HighPerformance Carbohydrate Analysis Column incorporates the familiar chemistryand selectivity of propyl amine functionality bonded onto the highly efficientsupport of 4 micron Nova Pak silica. This material is packed into a new columnformat. The Waters stainless steel cartridge column provides the ability to use anintegrated guard column to prolong the life of the analytical column. This designalso lowers the column replacement cost by incorporating reusable end fittings.

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© 2000 Waters Corp


Alliance System Reproducibility

M in u tes2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

6.6

µ R

I U

nits

Analyte Ret Time%RSD

Peak Area%RSD

1 Fructose 0.045 0.372 Glucose 0.056 0.423 Sucrose 0.078 0.534 Maltose 0.098 1.065 Lactose 0.097 0.94

1

23

45

Five InjectionOverlay

0.05% Each Sugar

The Alliance HPLC System demonstrates excellent reproducibility for analysis ofmono/disaccharides based upon retention time and peak area %RSD (percentrelative standard deviation).

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Response Linearity and Precision

Fructose Glucose Sucrose Maltose Lactose

Linearity, r2 0.9998 0.9981 0.9999 0.9999 0.9997

0.50% 0.49 0.49 0.56 0.53 0.98

0.25% 0.15 0.17 0.31 0.35 1.27

0.10% 0.56 0.65 0.68 0.43 0.82

0.05% 0.37 0.42 0.53 1.06 0.94

0.01% 1.87 6.46 2.44 4.31 6.68

0.005% 9.65 10.56 8.71 7.23 10.970. 005%

Data as Peak Area %RSD over 5 Injections

% S

uga

r C

onc

entr

atio

n

Excellent linearity and precision is also obtained. The precision decreases at the0.005% sugar concentration because we are approaching the method detection limitmaking it more difficult to integrate the smaller peaks. This is demonstrated in thenext slide.

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© 2000 Waters Corp

Minutes2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00


Low Concentration Detection

Fructose

Glucose

SucroseMaltose

Lactose

0.001% Sugar200 ng each

0.005% Sugar1 µg each

0.8

µ R

I U

nits

The method detection limit for the mono/disaccharides is 0.0001% or 200 ng ofeach sugar on column. However, note that labeling is not required until the levelsare quite high (0.5g total sugar per serving size).

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Sample Preparation

! Prepare approximately a 1% Solution of the foodmatrix• Add 80 mL of Warm (80°C) DI Water

• Sonicate for 15 Minutes

• Cool and Dilute to 100 mL

• Pass through a C18 Sep-Pak Cartridge to removelipid, protein, and suspended solids

• Use Filtrate for Analysis

Start with a sample concentration of 1 gram to 100 mL.

This is the sample preparation procedure utilized for all of the food products shownin this section.

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© 2000 Waters Corp

M in u tes2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00

6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00


1

2

3

4Unk

Unk

FiveInjectionOverlay

1.09g Honey / 100 mL

1 Fructose = 35.30 ± 0.02%2 Glucose = 31.34 ± 0.02%3 Sucrose = 0.46 ± 0.01%4 Maltose = 1.26 ± 0.02%

3.5

µ R

I U

nits

1.2

µ R

I U

nits

The following slides demonstrate the different sugar profiles obtained from fourfood products. Approximately 300 injections were performed on the same columnto demonstrate its ruggedness and reproducibility.

Natural honey contains high concentrations of fructose and glucose. Adulteration ofhoney can be determined by monitoring the fructose/glucose ratio. High fructosecorn syrup can be added to provide sweetness at less expense.

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© 2000 Waters Corp

Minutes2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

Analysis of Mono/DiSaccharideWaters Carbohydrate Analysis Column

16.

4 µ

RI U

nits

Milk Chocolate Candy Bar0.552g / 100 mL

1 Sucrose = 44.28 ± 0.07%2 Lactose = 7.43 ± 0.01%

ThreeInjectionOverlay

1

2

Sucrose and lactose (high in calories) are added to milk chocolate to make it sweet.

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© 2000 Waters Corp

Minutes2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00


Powdered Instant Breakfast Drink1.003 g / 100 mL

1

2

3

4

Five InjectionOverlay

1 Glucose = 0.54 ± 0.02%2 Sucrose = 30.35 ± 0.08%3 Maltose = 0.79 ± 0.02%4 Lactose = 23.43 ± 0.05%

20 µ

RI U

nit

s

This breakfast drink is marketed as providing high energy and nutritional value.Note that it also contains high levels of sucrose and maltose. Therefore, it is alsohigh in calories!

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© 2000 Waters Corp

Minutes2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00


FiveInjectionOverlay

1

2

3

4

1 Fructose = 2.66 ± 0.02%2 Glucose = 0.10 ± 0.001%3 Sucrose = 3.70 ± 0.03%4 Lactose = 3.51 ± 0.03%

Liquid Diet Breakfast Drink0.998 g / 100 mL

3.2

µ R

I U

nit

s

Note the contrast in this diet breakfast drink. This product contains significantlylower concentrations of sucrose and lactose so it also has a lot less calories! Lessexpensive high fructose corn syrup is added to make the product taste good.

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© 2000 Waters Corp


! Waters Carbohydrate Analysis Column is a 10 µmSilica Based Propyl Amine Column

! Current AOAC LC Methods• 977.20 Honey

• 980.13 Chocolate

• 982.14 Presweetened Cereal

• 984.17 Licorice Extracts

• 984.22 Purity of Lactose

Currently there are official AOAC methods for these analytes which incorporatesHPLC. It should be noted that they all use propyl amine chemistry bonded to silicastationary phases and that they are very specific to the matrix. Also note that thenewest of these methods is from 1984 and are still viable method today (2nd and3rd digits of the method number represent the year of acceptance). The reasons forthe matrix specificity are sample preparation issues and potential matrixinterference issues. Both of these issues can be minimized using the columnchemistry from Waters.

There are a variety of separation mechanisms and chemistries for the HPLCdetermination of sugars. Anion exchange, cation exchange, liquid/liquid partitionand size exclusion represent a few useful chemistries. However, with the five "foodsugars" commonly analyzed by food chemists, the propyl amine chemistry providesthe desired separation.

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© 2000 Waters Corp

Determination of Endocrine Disruptors inFood, Soil and Water

Using Novel Solid-Phase Extraction Sorbents

Michael S. YoungChromatography Chemistry Division

Waters Corporation

Introduction to the analysis of Endocrine Disruptors by HPLC using Oasis HLBcartridges for sample preparation.

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© 2000 Waters Corp

Endocrine Disruptors

Chemicals that affect the balance of normalhormonal functions in animals

• Pharmaceuticals such as diethylstilbestrol

• Natural products such as sterols found in somefoods

→ Environmental endocrine disruptors (EEDs)

The effect of endocrine-disrupting chemicals (EDCs) on human health and wild lifeis receiving growing attention from the scientific community, regulatory agencies,and the general public. The endocrine system includes the thyroid and reproductivesystems. Exposure to EDCs can mimic or interfere with naturally occurringhormones responsible for regulating reproductive and developmental bodilyprocesses.

Many endocrine disrupting chemicals such as pharmaceutical estrogens arefundamental to healthy lifestyles.

Naturally occurring EDCs such as phytoestrogens and isoflavanoids found in plantscan also be healthful.

Today we will focus on environmental endocrine distruptors that possibly causenegative health effects.

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Goal for This Study

To develop a solid-phase extraction (SPE)procedure which will give good results fordetermination of the majority of organic EEDs.

– The procedure must be compatible with both GC and LCtechniques

– The procedure must be applicable to polar and non-polartypes of compounds

– The procedure should be applicable to water, food andsoil

Objectives of this study

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© 2000 Waters Corp

Environmental Endocrine Disruptors(Catagories)

• Biocides (i.e. tributyl tin)

• Insecticides (i.e. DDT)

• Herbicides (i.e. atrazine)

• Nematocides (i.e. aldicarb)

• Fungicides (i.e. benomyl)

• Industrial Chemicals (i.e. bisphenol A)

• Metals (i.e. cadmium)

Here is a list of catagories of EEDs with an example of an environmental endocrinedisrupting chemical for each type.

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© 2000 Waters Corp

Endocrine DisruptorsEnvironmental Sources and Exposure

Modes• Bisphenol A - monomer for polycarbonate plastics

• Phthalate Esters - plasticizers– ingestion of leachate from plastic food packaging and plasticware

– ingestion of contaminated water

• Nonylphenol - degradation product of ethoxylated alkylbenzene non-ionic surfactants– ingestion of contaminated water

– direct exposure to the surfactants

• Pesticides– Ingestion of contaminated water, food, beverages

Examples of suspect endocrine disrupting chemicals and how we are exposed tothem.

Bisphenol A is commonly used in making polycarbonate plastics.

Phthlate esters are plasticzers used in the manufacuring process of plastics to makeit more flexible for molding machines. However, the plasticzers are easilyextracted from the finnished product such as baby teething rings.

Nonylphenol polyethoxylates were used as detergent additives in Switzerland

and Germany until their ban in 1986.

Pesticides are spayed on crops to protect them but also result in residues found infood and ground water.

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© 2000 Waters Corp

● Provides wetting properties● Reduces contact angle with

water● Provides enhanced retention for

polars

● Provides reversed-phaseproperty for analyte retention

Hydrophilicmonomer

Lipophilicmonomer

NO

Oasis® HLB SorbentA Hydrophilic-Lipophilic Balanced Copolymer

For sample preparation using solid-phase extraction, Oasis HLB is a more effectivereversed-phase sorbent for polar and non-polar analytes.

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© 2000 Waters Corp

SPE Method for Endocrine Disruptorsconditions for 6 cc, 200 mg Oasis® HLB cartridge

Condition/Equilibrate3 mL solvent*/3 mL methanol/3 mL water

Loadup to 500 mL sample

Wash3 mL 5% methanol in water

Elute6 mL 10%methanol/ MTBEt*

Prepare Sample adjust to pH 3

For LC analysis, exchange to acetonitrile, then adjust to 1 mL

For GC analysis, dry extractover Na2SO4, then adjust to 1 mL

Here is the universal sample preparation procedure using the Oasis HLB cartridgefor EEDs. Note that the sample extract can be analyized by GC and LC.

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© 2000 Waters Corp

• Use 200 mg Oasis® HLB Cartridge

• Use 250 - 1000 mL of sample for water analysis

• Use 40 - 200 mL of sample for beverage analysis

• Use an elution solvent which is compatible with GC but isvolatile and simple to remove by evaporation

• For LC, exchange the volatile solvent for an LC solventcompatible with mobile phase

• For GC, dry the extract and adjust final volume

Example: nonylphenol is too non-polar for elution withmethanol. Use 10 % methanol in MTBE for elution andexchange to acetonitrile for LC.

SPE Method for Endocrine Disruptors(Part 1. Liquid Samples)

Oasis sample preparation protocol for liquid samples.

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© 2000 Waters Corp

LC Determination of Endocrine DisruptorPhenols in Tap Water

Column: SymmetryShield™ RP18Mobile Phase: A: pH 3.0 phosphate buffer (15 mM)

B: acetonitrileGradient: 60% A initial, then linear

gradient to 100 % B in 20 minFlow Rate: 1.0 mL/minDetection: UV @ 225 nm (0.02 AUFS)Injection: 75 µL

1

2

1: bisphenol A, 240 ng/L2: nonylphenol (isomer mix), 1.2 µg/mL(estimated concentration of para n-nonyl isomer,100 ng/L)

spiked sample

non-spiked sample

Minutes0 10 20

Analysis of bisphenol A and nonylphenol in drinking water using the AllianceHPLC system with the Waters 996 PDA and Millennium32.

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© 2000 Waters Corp

Results are given as % recovery with % RSD in parenthesis

Level 1 Level 2 Level 3 Level 4 5 replicates 5 replicates 5 replicates 5 replicates

Compound

Bisphenol A 101 (17) 97.1 (2.9) 95.4 (1.0) 97.4 (1.2)

Nonylphenol n.a.* 82.9 (6.0) 80.0 (3.1) 78.0 (4.2)

Results, EED Phenols in Tap Water

Spike levels: bisphenol A - 0.24, 0.80, 3.2, 20 µg/Lnonylphenol - 1.2*, 4.0, 16, 100 µg/L

Estimated LOQs: bisphenol A - 0.1 µg/Lnonylphenol (mixed isomers) - 2.5 µg/L

* level 1 for nonylphenol, blank value greater than 50 % of spike level

Recovery results are excellent for bisphenol A and nonylphenol at four spike levelsfollowing the Oasis HLB sample preparation procedure for EEDs described earlier.Note that the result for nonylphenol, at spike level one (1.2 µg/L), was notapplicable because of nonylphenol contamination in the blank sample at that level.

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© 2000 Waters Corp

100

1000

10000

100000

100 1000 10000 100000

reco

very

leve

l, n

g/L

spike level, ng/L

BPA log/log plot

slope - 1.01corr. - 0.9995int. - 0.97

Recovery of Bisphenol A From Tap Water

Excellent recovery of bisphenol A in drinking water is also demonstrated using alog/log plot.

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© 2000 Waters Corp

LC Analysis of Phthalates in Tap Water

1. dimethyphthalate2. diethylphthalate3. ISTD (difluorobiphenyl)4. benzylbutylphthalate5. dibutylphthalate6. bis(ethylhexyl)phthalate7. dioctylphthalate

1

2

3

4 56

7

blank

Minutes0 10 20

HPLCColumn: SymmetryShield™ RP8Mobile Phase: A: water

B: AcetonitrileGradient: 50% B linear to 100% B in 10 minFlow Rate: 0.8 mL/minDetection: UV @ 196nm (0.03 AUFS)Injection: 20 µLSample: 150 mL of surface water

spiked @ 4 µg/L

Analysis of phthalates in drinking water using the Alliance HPLC system with theWaters 996 PDA and Millennium32.

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© 2000 Waters Corp

Results are given as % recovery with % RSD in parenthesis

4 µg/L 16 µg/L 5 replicates 5 replicates

Compound

dimethyl phthalate n.d. 87.2 (3.2)diethyl phthalate 97.4 (3.8) 92.3 (2.1)benzylbutyl phthalate 88.7 (14) 82.2 (1.6)dibutyl phthalate 88.0 (17) 87.7 (2.2)bis(ethylhexyl)phthalate 66.9 (22)** 72.1 (5.5)dioctyl phthalate 64.9 (22) 70.6 (5.6)

bis(ethylhexyl)adipate‡ 74.9 (22) 72.0 (4.2)

Results, Phthalates in Tap Water

n.d blank value greater than 50 % of spike level** blank value greater than 20 % of spike level ‡ results by GC/FID

Recovery results are excellent for the phthalates at two spike levels following theOasis HLB sample preparation procedure for EEDs described earlier. Note that theresult for dimethyl phthalate, at spike level 4 µg/L, was not applicable because ofdimethyl phthalate contamination in the blank sample at that level.

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© 2000 Waters Corp

Note: For LC analysis of fruit samples, the SPE eluent wascleaned up on aminopropyl silica. For GC fruit samples andall soil samples, no cleanup of SPE eluent was employed.

• Extract the sample (5-10 gm) with 20-40 mL acetonitrile orother water miscible solvent

• If desired, concentrate the organic extract by evaporation

• Dilute the organic extract with pure water (85-95% water)

• Perform SPE using the EED procedure– cleanup with normal-phase sorbents as necessary

SPE Method for Endocrine Disruptors(Part 2. Fruit and Soil)

Sample preparation protocol for EEDs in solid samples using Oasis HLB cartridgeand amino propyl Sep-Pak cartridge.

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© 2000 Waters Corp

LC Analysis of PearA Comparison of Extraction Procedures

acetonitrile extractdilute with water

Oasis® HLB extraction Sep-Pak® aminopropyl

cleanup

acetonitrile extractno cleanup

acetonitrile extractdilute with water

Oasis® HLB extraction

The benefits of solid phase extraction for sample cleanup are demonstrated in thiscomparison of extraction procedures for pears.

The upper chromatogram shows a separation of the pear sample after just extractingwith acetonitrile. Note the huge interference at the beginning of the chromatogram.

The middle chromatogram shows a separation of the same sample after extractionusing the Oasis HLB cartridge. Note the emergence of peaks that were previouslymasked by interferences in the sample matrix.

Finally, the lower chromatogram displays a “clean” separation of the pear sampleafter solid phase extraction using an Oasis HLB cartridge followed by an aminopropyl Sep-Pak cartridge. The amino propyl Sep-Pak cartridge also removed thecolor from the pear extract leaving a clear solution.

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© 2000 Waters Corp

Determination Polar EEDs in Pear100 ppb spike level

HPLCColumn: Symmetry™ C18, 3.9 x 150 mmMobile Phase: A: 10 mM phosphate pH 6.8

B: methanol Gradient: 40% B linear to 100% B in 20 minFlow Rate: 1.0 mL/minDetection: PDA (225 nm extracted, 0.1 AUFS )Injection: 100 µLSample: 10 g pear extracted with 25 mL

acetonitrile

12 3

4

1. benomyl2. carbaryl3. atrazine4. bisphenol A

% RECOVERY benomyl (interference)

carbaryl 99 + 4

atrazine 95 + 1

bisphenol A 86 + 2

Minutes0 5 10

sample

blank

Here are the chromatographic conditions for the separation of four polar compoundsthat are suspect environmental endocrine disruptors. This separation was performedon the Alliance HPLC System with the Waters 996 PDA and Millennium32.

The pear sample was spiked at the 100 ppb level with the four analytes andprepared using the SPE protocol described earlier for fruit samples.

Comparison of the spiked sample and blank shows a sample matrix interferencewhere the benomyl peak elutes. Note that the chromatogram was extracted at 225nm making it difficult to identify and quantitate benomyl. However, carbaryl,atrazine, and bisphenol A are easily detected and quantitated at that wavelength.

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© 2000 Waters Corp

Determination of Benomyl andBisphenol A at 283 nm

100 ppb spike level in pear


HPLCSame 3-D chromatogram as last slide

Detection: PDA (283 nm extracted)

% RECOVERY benomyl 65 + 10

bisphenol A 83 + 21

23 4

Minutes0 5 10

sample

blank

One of the advantages of a PDA detector is the ability to extract the chromatogramat different wavelengths. This slide shows the same sample extracted at 283 nm.At this wavelength, there is no interference for benomyl.

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© 2000 Waters Corp

Determination of Polar EEDs in Soil50 ppb spike level

HPLCColumn: Symmetry™ C18, 3.9 x 150 mmMobile Phase: A: 10 mM phosphate pH 6.8

B: methanol Gradient: 40% B linear to 100% B in 20 minFlow Rate: 1.0 mL/minDetection: PDA (225 nm extracted, 0.04 AUFS)Injection: 100 µLSample: 10 g soil extracted with 25 mL

acetonitrile

1

2 3

4


% RECOVERY + % RSD benomyl 62 + 6

carbaryl 91 + 4

atrazine 84 + 5

bisphenol A 78 + 6

( n = 5)

Minutes0 5 10

sample

blank

Under the same chromatographic conditions, a commercial potting soil was spikedat the 50 pp. level with the same analytes and prepared using the samplepreparation protocol for soil described in a previous slide.

Comparison of the spiked sample and blank demonstrates excellent detection andquantitation of the four analytes.

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© 2000 Waters Corp

ΙΙΙΙ PAH Analysis by HPLC Using PDA &Fluorescence Detection

ΙΙΙΙΙΙΙΙ Carbamate Analysis by HPLC/PCFD

Mark Benvenuti

Market Development

Waters Corporation

Introduction to the analysis of Polynuclear Aromatic Hydrocarbons (PAHs) byHPLC and Carbamates by HPLC with Post-Column Fluorescence Detection(PCFD).

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© 2000 Waters Corp

PAH Analysis with Alliance System and PDA Using aBinary Gradient

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

Minutes4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

1- Naphthalene - 20 ppm2- Acenaphthylene- 40 ppm3- Acenaphthene- 20 ppm4- Fluorene- 4 ppm5- Phenanthrene- 2 ppm6- Anthracene- 2 ppm7- Fluoranthene- 4 ppm8- Pyrene- 2 ppm9- Benzo(a)anthracene- 2 ppm10- Chrysene- 2 ppm11- Benzo(b)fluoranthene- 4 ppm12- Benzo(k)fluoranthene- 2 ppm13- Benzo(a)pyrene- 2 ppm14- Dibenzo(a,h)anthracene- 4 ppm15- Benzo(g,h,I)perylene-4 ppm16- Indeno(1,2,3-cd)pyrene-2 ppm

Column- HibarRT 125-4LiChrosphere PAHEluent A: WaterEluent B: AcetonitrileGradient: Linear A to B11 minutes, Hold 10 minutesBack to initial conditionsFlow Rate: 1.0 ml/minInjection: 20ul

UV@254nm

1

2

3

4

5

6

7

8

9

10

11

1213

14

1516

PAHs are among the most frequently analyzed organic pollutants in the world. Anew column chemistry available from Waters (part #PSL 000329) displays baselineresolution of 16 PAHs, listed in EPA Methods 550.1 and 8310, in less than twentyminutes using a water/acetonitrile gradient. The chromatogram was extracted at254 nm using the 996 PDA detector.

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© 2000 Waters Corp

PAH Analysis:PDA Spectrum Index Plot

nm

200.00

220.00

240.00

260.00

280.00

300.00

202.2

222.1

256.3

301.314.87

SI 11

237.4

307.215.55SI 12

226.8

264.5

296.5

16.19SI 13

1- Benzo(b)fluoranthene2- Benzo(k)fluoranthene3- Benzo(b)pyrene

AU

0.00

0.02

0.04

0.06

0.08

Minutes14.60 15.00 15.40 15.80 16.20 16.60

1

23

The Waters Millennium32 PDA software allows you to conveniently extract thepeak apex spectrum and display it over the chromatogram.

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© 2000 Waters Corp

Purity Plot for Chrysene

Purity angle of0.249 is les thanthreshhold of 1.034

AU

Degrees

0 .00

0.01

0.02

0.03

0.04

0.05

0.06

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

M inutes13.40 13.45 13.50 13.55 13.60 13.65 13.70 13.75 13.80 13.85 13.90

P urityN oise+ S olvent (1.00)

---------- Absorbance

---------- Threshold Angle

---------- Purity Angle

---------- Baseline

Millennium32 software allows you to easily check the purity of specific peaks bothvisually and mathematically.

In this case, the chromatographic peak chrysene has a purity angle which is locatedbelow the threshold angle indicating a homogeneous peak. This is confirmedmathematically for chrysene, since the purity angle of 0.249 is less than thethreshold angle of 1.034

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© 2000 Waters Corp

PDA Identification of ChryseneA

U

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

nm200.00 210.00 220.00 230.00 240.00 250.00 260.00 270.00 280.00 290.00 300.00 310.00

------------- sample spectrum

------------- library spectrum

Each peak in a sample can be automatically identified using the Library Matchfunction of the Millennium32 software . Library Match mathematically comparesunknown peaks to reference spectra stored in a user built library.

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© 2000 Waters Corp

Library Match Triple Plot for ChryseneA

U

0.05

0.10

200.00 210.00 220.00 230.00 240.00 250.00 260.00 270.00 280.00 290.00 300.00 310.00

220.9

266.9

307.2

Chrysene

AU

0.05

0.10

200.00 210.00 220.00 230.00 240.00 250.00 260.00 270.00 280.00 290.00 300.00 310.00

220.9

266.9

306.0

Match #1 Angle 0.465 Chrysene

AU

-0.005

0.000

nm200.00 210.00 220.00 230.00 240.00 250.00 260.00 270.00 280.00 290.00 300.00 310.00

229.2 269.3 312.0

Difference

Multiple plot spectral comparisons and difference plots allow you to visuallyevaluate the quality of the library match and display where a spectral differencemay have occurred.

42

© 2000 Waters Corp

PAH Analysis with Alliance System and FluorescenceUsing a Binary Gradient

1- Naphthalene - 2000 ppb2- Acenaphthylene- 4000 ppb3- Acenaphthene- 2000ppb4- Fluorene- 400ppb5- Phenanthrene- 200ppb6- Anthracene- 200ppb7- Fluoranthene- 400ppb8- Pyrene- 200ppb9- Benzo(a)anthracene- 200ppb10- Chrysene- 200ppb11- Benzo(b)fluoranthene- 400ppb12- Benzo(k)fluoranthene- 200ppb13- Benzo(a)pyrene- 200ppb14- Dibenzo(a,h)anthracene- 400ppb15- Benzo(g,h,I)perylene-400ppb16- Indeno(1,2,3-cd)pyrene-200ppb


mV

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The EPA approved HPLC methods require both UV absorbance and fluorescencedetection. The chromatograms shown in this section were obtained using and anAlliance HPLC System with 996 PDA and 474 fluorescence detectors connected inseries. Using this configuration, both UV and fluorescence chromatograms areacquired from the same injection.

Greater sensitivity is achieved for PAHs with fluorescence detection. Note thatacenapthene does not fluoresce, necessitating UV detection..

43

© 2000 Waters Corp

Sub PPB PAH Analysis with Alliance System andFluorescence Using a Binary Gradient

1- Benzo(a)anthracene- 0.2ppb2- Chrysene- 0.2ppb3- Benzo(b)fluoranthene- 0.4ppb4- Benzo(k)fluoranthene- 0.2ppb5*- Benzo(a)pyrene- 0.2ppb6- Dibenzo(a,h)anthracene- 0.4ppb7- Benzo(g,h,I)perylene-0.4ppb8- Indeno(1,2,3-cd)pyrene-0.2ppb


mV

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M inutes12.5 0 13.0 0 13.5 0 14.0 0 14.5 0 15.0 0 15.5 0 16.0 0 16.5 0 17.0 0 17.5 0 18.0 0 18.5 0 19.0 0 19.5 0

1

2

3

4

5*

6 7 8

5* Regulated Compound

This chromatogram demonstrates the ability to detect PAHs at 0.2-0.4 ppb by directaqueous injection using fluorescence detection.

The maximum contaminant level (MCL) for benzo(a)pyrene, a regulated compoundin the US, is 0.2 ppb.

44

© 2000 Waters Corp

Alliance System for CarbamateAnalysis

Waters Alliance System for Carbamate Analysis consists of a 2690 SeparationsModule with column heater assembly, a Post Column Reaction Module, aTemperature Control Module, two Reagent Managers, a 474 Scanning FluorescenceDetector, Millenium32 Chromatography Software, and Waters Carbamate Analysiscolumn.

Carbamates are systemic pesticides used to protect a wide variety of crops.

N-methylcarbamates are detected as a highly fluorescent isoindole, which is formedin a two-step, post-column derivatization reaction. The first step involveshydrolysis with aqueous sodium hydroxide. The reaction is performed at elevatedtemperature in a proprietary knitted RXN 1000 reaction coil located in the PostColumn Reaction Module. It is followed by reaction with o-phthalaldehyde (OPA)in the presence of 2-mercaptoethanol to produce the isoindole. A patentedcountercurrent heat exchanger (CHEX) preheats the incoming solvents and coolsthe exiting solvents to ambient temperature prior to exiting the Post ColumnReaction Module for the fluorescence detector.

45

© 2000 Waters Corp

Alliance System forCarbamate Analysis Reproducibility

mV

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Seven injections - 25ppb leveleach analyteRetention time < 0.17% RSDPeak Area < 1.2% RSDexcept 1-Naphthol 1-Aldicarb Sulfoxide

2-Aldicarb Sulfone3-Oxamyl4-Methomyl5-3-Hydroxycarbofuran6-Aldicarb7-Propoxur8-Carbofuran9-Carbaryl10-1-Naphthol11-Methiocarb

12 3 4

5

6

7 8

9

10

11

The retention time data (<0.17%) and peak area (<1.2% RSD) testify to theexcellent reproducibility of the Alliance System for Carbamate Analysis.

46

© 2000 Waters Corp

Carbamate Detection LimitUsing PCFD Method

Method Detection Limits Using EPA Definition 1 Aldicarb Sulfoxide 0.06 ppb 6 Aldicarb 0.09 ppb 2 Aldicarb Sulfone 0.09 ppb 7 Propoxur 0.12 ppb 3 Oxamyl 0.15 ppb 8 Carbofuran 0.12 ppb 4 Methomyl 0.06 ppb 9 Carbaryl 0.10 ppb 5 3-HydroxyCarbofuran 0.05 ppb 10 Methiocarb 0.16 ppb

Based upon std dev of 7 replicate injections; MDL = 3.14 x Std Dev

Blank Gradient

0.00 5.00 10.00 15.00 20.00Minutes

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1 23 4

6 7 8

9

10

0.2 ppb Standards

0.5 ppb Standards

1.5

mV

The technology contained in the Waters Alliance System for carbamate Analysisenables the detection of significantly lower levels of carbamates. As illustrated inthis slide, superior resolution and detection of carbamates at 0.2 ppb is easilyaccomplished.

Special thanks to Linda Henry (American Water Works Service Company, Inc.Belleville, IL) for providing these data.

47

© 2000 Waters Corp

LC Analysis of Carbamates in Drinking WaterSpike Level 50 ng/L, SPE Method for EEDs

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1. Aldicarb Sulfoxide2. Aldicarb Sulfone3. Oxamyl4. Methomyl5. 3-Hydroxycarbofuran6. Aldicarb7. Propoxur8. Carbofuran9. Carbaryl10. Methiocarb11. ISTD (BDMC)

Minutes2.00 8.00 14.00 20.00 26.00

Sensitivity for carbamates can be increased even further by incorporating solidphase extraction. Shown here is a drinking water sample from Milford, MA. 200mL of water was spiked at 50 ng/L (50 parts per trillion) and extracted on the OasisHLB cartridge using the EED sample preparation procedure for liquid samplesdescribed earlier.

48

© 2000 Waters Corp

50 ng/L 500 ng/L 5 replicates 5 replicates

Compound % recovery % RSD % recovery % RSD

aldicarb sulfoxide 45.7 (5.1) 54.7 (0.5)aldicarb sulfone 101 (3.6) 98.7 (4.0)oxamyl 122 (18) 90.8 (7.0)methomyl 100 (3.2) 99.9 (6.4)3-hydroxycarbofuran 111 (6.5) 98.7 (2.3)aldicarb 104 (5.8) 90.7 (9.3)propoxur 99.9 (5.1) 97.5 (5.6)

carbofuran 104 (7.9) 97.2 (4.7)carbaryl 122 (11) 89.6 (2.2)methiocarb 120 (14) 91.6 (2.2)

Results, Carbamates in Tap Water

Recovery results for carbamates at two spike levels are excellent.

49

© 2000 Waters Corp

Method for Carbamates in Cider

• 40 mL sample adjust to pH 5 - 6

• Centrifuge at 8000 x g for 10 minutes

• Perform SPE*

* Use the General Endocrine Disruptor SPE Protocol:

6 cc, 200 mg Oasis® HLB cartridgeElute with 10% methanol/MTBE

If desired, the extract can be cleaned upusing Sep-Pak® aminopropyl cartridges

The next experiment was a more complex sample.

Fresh apple cider was obtained from a cider mill located in western Massachusetts.It had a brown color and lots of sediment. The sample preparation required using acentrifuge to remove undesirable material from the sample followed by solid phaseextraction using an Oasis HLB cartridge followed by an amino propyl Sep-Pakcartridge. The amino propyl Sep-Pak cartridge also removed the color from theapple cider extract leaving a clear solution. Removal of the color preventedpossible chemical contamination of the analytical column but did not affect thechromatography.

50

© 2000 Waters Corp

Minutes0 15 30

LC Analysis of Carbamates in Apple Cider

HPLCColumn: Waters™ Carbamate Analysis

3.9 x 150 mmMobile Phase: A: Water

B: MethanolC: Acetonitrile

Ternary Non-Linear gradientFlow Rate: 1.5 mL/minDetection: Post Column Derivatization

Fluorescence, 339 nm (ex)445 nm (em)

Injection: 75 µLSample: 40 mL of cider spiked @ 2.5 µg/L

1. Aldicarb Sulfone2. Oxamyl3. Methomyl4. 3-Hydroxycarbofuran5. Aldicarb6. Propoxur7. Carbofuran8. Carbaryl9. Methiocarb10. ISTD (BDMC)

12 3

45

6 7

8

9

10

nonspiked sample

The prepared apple cider extracts were then injected into the Alliance System forCarbamate Analysis under the chromatograhic conditions described in the slide.

Comparison of the spiked sample (2.5 ppb) and non-spiked sample demonstrateinterference free chromatographic separations of the apple cider extracts. Thecarbamates are easily detected and quantitated at the spike level.

Note in the lower chromatogram of the nonspiked sample that carbaryl (peak 8) wasactually found in the cider. There is also two small peaks that seem to have thesame retention time as peaks 2 and 3 (oxamyl and methomyl) in the upperchromatogram. This is an example of where LC/MS can be utilized to confirm theidentity of those two peaks.

51

© 2000 Waters Corp

2.5 µg/L 5 replicates

Compound % Recovery % RSD Blank Corrected

aldicarb sulfoxide not recoveredaldicarb sulfone 33.6 (14) 30oxamyl 120 (15) 80methomyl 82.6 (9.9) 603-hydroxycarbofuran 142 (4.9) 120aldicarb 116 (1.7) 110propoxur 116 (4.2) 110carbofuran 112 (4.2) 110

carbaryl 582 140methiocarb 126 (7.6) 120

Results, Carbamates in Apple Cider

Recovery results for carbamates in apple cider using the sample preparationprotocol described previously are good.

52

© 2000 Waters Corp

Glyphosate Analysis Using theAlliance System for Carbamates

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Seven replicate injections, 100 ppb level

Retention Time < 0.1 % RSD

Peak Area < 0.7 % RSD

1

2

1- Glyphosate

2- AMPA

Glyphosate is a widely used agricultural and domestic household herbicide.Glyphosate and its major metabolite amino-methylphosphonic acid (AMPA)analyzed with the Alliance System for Carbamate Analysis simply by changing thecolumn, mobile phase, and post column reagents.

Using the Waters Ion Exclusion column and dilute phosphoric acid, analysis timeof glyphosate and AMPA is 30 minutes.

Glyphosate is detected as a highly fluorescent isoindole, which is formed in a two-step, post-column derivatization reaction. The first step involves oxidation withhypochlorite (Clorox). The reaction is performed at elevated temperature in aproprietary knitted RXN 1000 reaction coil located in the Post Column ReactionModule. It is followed by reaction with o-phthalaldehyde (OPA) in the presence of2-mercaptoethanol to produce the isoindole. A patented countercurrent heatexchanger (CHEX) preheats the incoming solvents and cools the exiting solvents toambient temperature prior to exiting the Post Column Reaction Module for thefluorescence detector (excitation:339nm, emission:445 nm).

53

© 2000 Waters Corp

Glyphosate Analysis using the Alliance System for Carbamates

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Seven replicate injections, 10 ppb levelRetention Time < 0.1% RSDPeak Area < 3.5% RSD

1

2

1- Glyphosate2- AMPA

Note the excellent sensitivity and reproducibility for this analysis.

54

© 2000 Waters Corp

Challenges in Using LC/MS forCarbamate Analysis

• Developing appropriate chromatographic conditions.

• Using standards to determine characteristic retention timesand mass spectral behavior.

• Achieving acceptable limits of detection and quantitation toinsure reliable pesticide screening.

• Achieving the same limits of detection and quantificationwith actual samples in matrix.

Using LC/MS for carbamate analysis imposes specific requirements.

Unlike HPLC/UV detection, LC/MS response places some restraints on HPLCmobile phase composition and flow rate. Transferring a method from HPLC toLC/MS requires changing the column dimension (1-3mm ID) and flow rate (0.1-0.5mL/min) to maintain good separation efficiency. One must chose mobile phasebuffers that are amenable to the mass spectrometer interface.

Unlike UV detection, mass spectrometer response is very much compounddependent. For maximum sensitivity and selectivity, the analyst will want todetermine the choice of ionization mode (ESI or APCI,) as well as ion source tuningconditions, gives the best response. While most instruments have some form of“Autotune” program for determining the optimum conditions, the best way to dothis is by introducing the target analyte into the source and manually optimizing thesource conditions for each interface. In practice, this is not as complicated as itsounds, and only requires about 2 minutes per compound.

55

© 2000 Waters Corp

Carbamates - Analysis byAPI LC/MS

Eric Block, John Van Antwerp

LC/MS

Waters Corporation

Introduction to carbamate analysis by Atmospheric Pressure Ionization (API)LC/MS.

56

© 2000 Waters Corp

Appropriate ChromatographicConditions

HPLC: Waters Alliance™ SystemColumn: Waters Symmetry™C18, 1.0 x150 mmTemperature: 35°CInjection Volume: 10µLMobile Phase:

A: 10% Methanol/10 mM NH4AcB: 90% Methanol/10 mM NH4Ac

Flow Rate: 75 µL/min

Gradient: Time %A %B Curve Initial 90 1010 10 90 612 10 90 6

Note that the chromatographic conditions are different from the HPLC post-columnderivatization method .

A Symmetry C18 with a 1.0 mm ID (inner diameter) was used.

An ammonium acetate buffer was added to the mobile phase in order to createbetter ionization for the mass spectrometer.

Optimum flow rate for the column dimension was 75 µL/min.

The gradient program for the HPLC post-column derivatization method is fairlyintricate, because fluorescence and UV detection are 2-dimensional. For goodquantitation, each peak must be baseline resolved from one another. Mass Spec, onthe other hand, adds the additional dimension of mass, allowing the massspectrometer to “resolve” co-eluting compounds on the basis of their molecularweight. For this reason, the simpler and faster gradient, shown here as used.

Note. Later work with spiked matrix used a 2.1 mm x 150 mm column. By simply scalingup the flow rate, the same chromatography could be obtained without the excessive delaytimes incurred with the microbore column.

57

© 2000 Waters Corp

Mass Spectrometer ConditionsInstrument: Waters ZMD ZSpray™ Mass Detector

Interface: Positive Electrospray (ESI+)

Scan Function: Multiple Selected-Ion Recording (SIR):

Time Cone Dwell Group mins. Compound Mass Voltage Time

1 0-9 Aldicarb Sulfoxide 207.1 18V 0.5 secsAldicarb Sulfone 223.2 25V 0.5 secsOxamyl 237.2 10V 0.5 secsMethomyl 163.2 15V 0.5 secs

2 9-11 3-OH Carbofuran 238.2 15V 1.5 secs3 10.5-12.5 Aldicarb 208.2 8V 1.5 secs4 11.5-14 Propoxur 210.2 18V 0.4 secs

Carbofuran 222.2 22V 0.4 secsCarbaryl 202.2 18V 0.4 secs

5 14-20 Methiocarb 226.2 19V 0.6 sec

MassLynx ™ Software

The ZMD mass detector can easily switch between positive and negative ionmonitoring. However, all of the analytes in this case worked best using theelectrospray interface in the positive ion mode. The ZMD operates in full scan orselected ion recording (SIR) acquisitions modes.

The best sensitivity for carbamates is obtained running in SIR mode. TheMassLynx software group function tells the mass spectrometer which ions tomonitor. This slide shows the acquisition program used for quantitative analysis. Aspecific ion is monitored for each analyte over its retention time window. As eachion is monitored, the mass spectrometer uses the optimum cone voltage setting forthat analyte, increasing sensitivity.

58

© 2000 Waters Corp

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00T im e0

100

%

12.51

9.95

6.67

6.05 7.3911.44

15.17

12.96

0.00 2.50 5.00 7.50 10.00T ime0

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%

1

100

%

4

100

%

1

100

%

B : A ldicarb Su lfon eC : O xamylD : MethomylE : 3-OH Carbofura nF : A ldicarbG : P ropoxurH: C arbofuranI : C arbarylJ : Meth iocarb

A : A ldicarb S ulfoxideCarbamates Full-Scan TIC

AB,C

D

E

F

G,H

IJ

Oxamyl [M+NH4]+1 = 237.2

Methomyl [M+H]+1 = 163.2

Aldicarb [M+H]+1 = 223.2Sulfone

Aldicarb [M+H]+1 = 207.1Sulfoxide

Selected Ion Recording

20 ng Each on-column

Minutes

This is a good illustration of how the mass spectrometer can resolve co-elutingpeaks. The large plot is the total ion chromatogram, which plots the sum of all ionsignal in each scan versus time. It is roughly equivalent to a UV or fluorescencedetector output. As can be seen, the early-eluting compounds are poorly resolvedand co-elute. However, since each mass acts as an independent data channel, byplotting masses specific to each compound (inset), the mass spectrometer is able toextract four discrete peaks. Since the masses are distinct for each compound, theycan be quantitated even though they co-elute.

Note: The poor peak shape of the first compound is a result of the fact that thesample was injected from a 100% organic solution. Subsequent work usedstandards/samples in more aqueous solutions, which improved peak shape of theearly-eluting components.

59

© 2000 Waters Corp

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00Time0

100%

0

100%

0

100%

0

100%

0

100%

9.68

6.94

6.27

6.07

5.28

2

100%

4

100%

-18

100%

1

100%

4

100%

15.02

12.83

12.36

12.31

11.23

Ald. Sulfoxide

Ald. Sulfone

Oxamyl

Methomyl

3-OH Carbofuran

Aldicarb

Propoxur

Carbofuran

Carbaryl

Methiocarb

Composite SIR Chromatograms of 10 CarbamatesSensitivity: 50 pg On-Column

s/n=6:1

s/n=80:1

This slide illustrates the sensitivity of the method. A mixture of all compounds (5pg/µL) was injected and analyzed. Displayed are all the chromatograms of all themonitored ions. This represents 50 pg (<500fmol) of each compound on-column.Signal to noise varied from 6:1 for methomyl, to approximately 80:1 for propoxur

60

© 2000 Waters Corp

m/z

[M+H]+

120 140 160 180 200 220 240 260 280 300 320 340 360 380 4000

100

%

163

185 [M+Na]+

Background-subtracted Full-scan ESI+ Spectrumof Methomyl

[M+K]+ [2M+H]+

[2M+Na]+

This slide shows the mass spectrum of methomyl acquired by the ZMD using theelectrospray interface in positive ion mode. The most intense ion is the [M+H] + ionat 161. However, several other ions are seen, including the sodium and potassiumadduct ions as well as the dimer and its adduct ion.

This is a good illustration of how instrument response is compound-dependent.

Note: These slides are from infusion of individual carbamate standards atconcentrations of approximately 20 ppm. Dimerization is not uncommon wheninfusing relatively high concentrations of sample.

61

© 2000 Waters Corp

120 140 160 180 200 220 240 260 280 300 320 340 360 380 400

m/z

0

100

%

237

223

242

278

[M+NH4]+

[M+Na]+

[M+H]+

Background-subtracted Full-scan ESI+ Spectrumof Oxamyl

For Oxamyl, the expected protonated molecular ion at m/z 200 is minisculecompared to the adduct ions. Clearly this compound is very ionophoric, and willscavenge any cations in solution. For this reason the mobile phase contained 10mM ammonium acetate in order to provide a constant level of ammonium cationsand the ammonium adduct at m/z 237 was monitored in subsequent quantitativeanalyses.

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© 2000 Waters Corp

120 140 160 180 200 220 240 260 280 300 320 340 360 380 400m/z

0

100

%

202

145

219

[M+H]+

[M+NH4]+

Background-subtracted Full-scan ESI+ Spectrumof Carbaryl

This slide shows the resulting ESI spectrum of Carbaryl. This compound gave asimpler spectrum where the protonated molecular ion at m/z 202 displayed thegreatest intensity. This ion would be monitored for quantitative studies.

63

© 2000 Waters Corp

Quantitation of Carbamates via LC/MS

• Once the retention times and mass spectral behavior ofcarbamates is known under appropriate chromatographicconditions, we are ready to quantitate.

• Must still achieve acceptable limits of detection andquantitation to insure reliable pesticide screening.

• Must still achieve the same limits of detection andquantitation with actual samples in matrix.

In order to quantitate, we must first evaluate the sensitivity, linearity, accuracy, andprecision of the method using standards. Then we can run actual samples.

64

© 2000 Waters Corp

0.0 200.0 400.0 600.0 800.0 1000.0ng/mL

0

Compound 4 name: methomyl (m/z 163)Coefficient of Determination: 0.995876Response type: External Std, AreaCurve type: Linear, Weighting: 1/x 5.96e6

Response

Methomyl Calibration Curve; 5-1000 ng/mL

Here is a representative calibration curve for methomyl.

65

© 2000 Waters Corp

Linearity and SensitivityLinearity was assessed from triplicate analysis of a series of calibration standards(5-1000 ng/mL). Instrumental LOD and LOQ were defined as 3X and 10X thestandard deviation of the calculated concentrations, respectively. LOD and LOQwere determined from 5 replicate injections of a standard mixture (50 pg eachanalyte on-column).

Coefficient of determination LOD* LOQ*

Aldicarb Sulfoxide 0.9969 8 26Aldicarb Sulfone 0.9982 18 61

Oxamyl 0.9990 7 22

Methomyl 0.9959 16 55

3-OH Carbofuran 0.9970 4 14

Aldicarb 0.9963 2 5

Propoxur 0.9967 17 55

Carbofuran 0.9981 9 30

Carbaryl 0.9994 3 11

Methiocarb 0.9995 4 14

* pg on-column

Linearity was determined over a 200-fold concentration range with very goodresults.

Note: Unlike UV detection, the mass spectrometer is inherently non-linear. Thedegree f non-linearity is compound dependent and also varies with analyteconcentration range and matrix effects. This as an inherent behavior of APIionization, and is not instrument specific. For this reason, weighting is often used.This is NOT CHEATING! Also note that, because linear regression used aweighting factor, the familiar term “R2” is replaced with statistically equivalent“coefficient of determination.”

The lowest standard was injected in replicate (n=5) in order to determine theinstrumental limits or detection (LOD) and quantitation (LOQ). This slide listsinstrumental sensitivity limits calculated for the amount of material injected on-column.

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© 2000 Waters Corp

Precision and AccuracyFive replicate injections of a 5 ng/mL standard solution were made. Precision isdefined as the percent coefficient of variation of the calculated concentrations.Accuracy is defined as the percent difference from theoretical of the meancalculated concentration.

Mean(ng/mL)

S.D. % C.V. % Diff.

Aldicarb Sulfoxide 7.48 0.259 3.5 49.6Aldicarb Sulfone 6.32 0.606 9.6 26.4Oxamyl 6.68 0.217 3.2 33.6Methomyl* 5.50 0.548 10.0 10.03-OH Carbofuran 6.80 0.141 2.1 36.0Aldicarb 7.36 0.055 0.7 47.2Propoxur 4.90 0.552 11.3 -2.0Carbofuran* 3.98 0.299 7.5 -20.5Carbaryl 6.26 0.114 1.8 25.2Methiocarb 5.70 0.141 2.5 14.0

*n=4

Linearity is nice so is sensitivity, but the bottom line is accuracy and precision.This slide presents the accuracy and precision results from replicate injections ofthe lowest standard. Precision (defined as % C.V.) and accuracy (defined as the %difference from theoretical of the mean) are quite good.

Note: These values are probably higher than what people are used to seeing withLC/UV methods, but it should be borne in mind that we are talking about very lowamounts (<500 fmol) on-column; levels that are unobtainable by UV detection.Obviously, precision and accuracy values are much better for higherconcentrations. This slide shows “worst case” data. Also, remember that unlike aUV detector, LC/MS is a very dynamic process. When working at the ragged edgeof concentration, precision and accuracy often fall off. Still, by LC/MC standards,these results are very respectable.

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© 2000 Waters Corp

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00

Time

1

100

%

S/N:PtP=22.84

Overlaid SIR Chromatograms of Aldicarb(50 pg on-column) and a Blank Injection

Blank

This Selected -Ion Recording chromatogram of Aldicarb represents 50 pg(<500fmol) on column. Signal to noise is 22.84 to 1.

68

© 2000 Waters Corp

But What About Real Samples?

Based on standards, we have demonstrated that our method provides adequatesensitivity, linearity, precision, and accuracy. Unfortunately, analytical chemistsare not paid to analyze standards. We have to look at real samples, which have atendency to be messy. A better test of this method would be to apply it to theanalysis of real samples. Let’s take a look at the results of some actual samples.

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© 2000 Waters Corp

SPE Recovery; 500 ppt in Tap Water

0 50 100

Ald.Sulfoxide

Ald. Sulfone

Oxamyl

Methomyl

3-OH Carbofuran

Aldicarb

Propoxur

Carbofuran

Carbaryl

MethiocarbC

om

po

un

d

% Recovery

LC/MS

LC/PCFD

The first matrix we looked at was water. Milford drinking water was spiked with acarbamate mixture at 500 ppt, and extracted and concentrated by SPE cleanup usingOasis HLB cartridges. The sample was analyzed by both traditional post-columnderivatization fluorescence detection (red) as well as by LC/MS (green), and therecoveries were compared.

As can be seen, there is good agreement between the two methods. The differencesfor aldicarb sulfoxide are believed to be due to sample degradation in the timeinterval between the two analyses.

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© 2000 Waters Corp

Carbamates LC/MSA comparison of results obtained from 500 ng/L spiked drinking water samples.Each extract was analyzed by LC/MS and also by LC with post-columnderivatization/fluorescence detection (PCFD). [% recovery (% RSD)]

Compound LC/MS LC/PCFDaldicarb sulfoxide 74.8 (19) 54.7 (0.5)aldicarb sulfone 88.7 (16) 98.7 (4.0)oxamyl 83.2 (18) 90.8 (7.0)methomyl 92.3 (8.0) 99.9 (6.4)3-hydroxycarbofuran 101 (8.6) 98.7 (2.3)aldicarb 79.4 (9.3) 90.7 (9.3)propoxur 103 (13) 97.5 (5.6)carbofuran 95.6 (7.5) 97.2 (4.7)carbaryl 97.7 (14) 89.6 (2.2)

methiocarb 81.2 (14) 91.6 (2.2)

Here is a comparison of the actual results.

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© 2000 Waters Corp

LC/PCFD Analysis of HarvestedBell Pepper Extract # 00329

mV

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Minutes4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

1

2

1- Methomyl 46.0 ppb

2- Propoxur 293.8 ppb *

* spiked in sample

Bell peppers are more complex than water. We were provided with several extractsfrom peppers with incurred (and therefore unknown) levels of carbamates exceptfor propoxur, which was spiked into all the samples.

The samples were analyzed by the traditional post-column derivatizationfluorescence detection method. Methomyl was detected in this sample.

72

© 2000 Waters Corp


Minutes

4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

1

2

3

1- Oxamyl- 32.4 ppb2- Propoxur- 280.6 ppb *3- Carbaryl- 14.2 ppb

* spiked in sample

mV

0.005.00

10.0015.0020.0025.0030.0035.0040.0045.0050.0055.00

Here is a chromatogram of a different sample where oxamyl and carbaryl weredetected.

73

© 2000 Waters Corp


mV

-5.000.005.00

10.0015.0020.0025.0030.0035.0040.0045.0050.0055.00

Minutes4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

1

2

3

1- Oxamyl- 54.1 ppb

2- Propoxur- 290.5 ppb *

3- Carbaryl- 136.8 ppb

* spiked in sample

Detection of carbamates in another sample of bell pepper.

74

© 2000 Waters Corp

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50

Time

1

100

%

1

100

%

1

100

%

5.23

5.26

2.97

1.33 1.90

Low Standard 200 pg on-column

Positive Pepper Sample 00329 approx. 47 pg on-column

Negative Pepper Sample 00340

LC/MS Analysis of Harvested BellPeppers: Methomyl, 2µL Injections

The same samples were also analyzed by LC/MS. Quantitation was done on thebasis of a 3-point standard. This slide compares the methomyl chromatogram fromthe low standard (top) with chromatograms from positive (middle) and negative(bottom) pepper extract. As may be seen from the extracts, the selectivity providedby LC/MS results in extremely clean baselines.

75

© 2000 Waters Corp

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50Time

4

100

%

4

100

%

4

100

%

4.14

4.11

Low Standard 240 pg on-column

Positive Pepper Sample 00340approx. 150 pg on-column

Negative Pepper Sample 00329

LC/MS Analysis of Harvested BellPeppers : Oxamyl, 2 µL Injections

These chromatograms compare a standard, positive, and negative sample foroxamyl.

76

© 2000 Waters Corp

LC/MS Analysis of Harvested BellPeppers : Carbaryl, 2µL Injections

10.60 10.80 11.00 11.20 11.40 11.60 11.80 12.00 12.20 12.40Time

6

100

%

6

100

%

6

100

%

Negative pepper Sample 0329

Positive pepper Sample 0336approx. 27 pg on-column

Low Standard120 pg on-column

One last slide showing carbaryl response.

77

© 2000 Waters Corp

Harvested Bell Pepper Samples

Sample HPLC LC/MS HPLC LC/MS HPLC LC/MS HPLC LC/MSID (ppb) (ppb) (ppb) (ppb) (ppb) (ppb) (ppb) (ppb)

329 46 46.5 - - - - 293.8 276.5

330 411.5 342.5 - - - - 319.7 323.5

332 32.5 40.5 - - - - 298.6 241.0

336 - - 32.4 48.0 14.2 13.5 280.6 263.5

340 - - 54.1 76.0 136.7 154.5 290.5 341.5

Methomyl Oxamyl Carbaryl Propoxur

These samples had incurred carbamate levels except for propoxur, which wasspiked into all the samples, and so the actual concentration was unknown, outside ofthe LC/MS determination. As an additional check, the samples were analyzed viathe traditional post-column derivatization fluorescence method, and the meandetermination of both methods were compared. As may be seen, there is goodagreement between the two methodologies.

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© 2000 Waters Corp

Confirmation/Screening

• “Three-ion rule” used to eliminate false positives

• In this example, “In-Source CID” was used togenerate fragment ions from the analyte(Methomyl)

• Relative abundance ofpseudomolecular/fragment ion ratios werecompared for standard and spiked spinachextracts (n=10)

The “3-ion rule” is a convention which arose from the early days of GC/MS. Inorder to provide confirmation and avoid false positives, three structurally significantions are monitored by SIR for each compound. Identification is consideredconfirmed if all three ions have the same retention time/chromatographic profileand their relative intensities match those of an authentic standard. Unfortunately,atmospheric-pressure ionization spectra typically do not yield abundant fragmentions as is the case for electron-impact

ionization used in GC/MS. However, fragmentation can be induced by increasingthe voltage on the sampling cone in a process known as “in-source CollisionallyInduced Dissociation.”

Note: CID fragmentation is extremely compound and tune-dependent; CID spectraare not nearly as reproducible as electron-impact spectra. Also, bear in mind thatas sampling cone voltage is increased to induce fragmentation, very often absolutesignal will decrease, meaning that the analyst will have to strike a balance betweenoverall sensitivity and confirmation.

79

© 2000 Waters Corp

50 60 70 80 90 100 110 120 130 140 150 160 170 180m/z

0

100

%

106

88

163

122

OH3 CS

CH3

N NHCH3

O882H

106

CID Fragmentation of Methomyl

[M+H]+

This is an abbreviated spectrum of methomyl under CID conditions. As may beseen, several structurally significant fragment ions are observed. The insert showsthe proposed identity of these fragments.

Note: The ion at m/z 122 cannot be explained by a simple bond cleavage andprobably arises from rearrangement during the CID process. Because of theunknown identity of this ion, it was not considered in the following slides.

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© 2000 Waters Corp

Methomyl Confirmation in Spinach

Ion m/z 88 Da 106 Da 163 Da 88 Da 106 Da 163 Da

% 100 51 34 100 55 34Relative 100 56 36 100 62 36Abundance 100 54 34 100 55 30

100 51 32 100 65 35100 55 31 100 63 36100 52 30 100 65 37100 54 30 100 69 38100 54 30 100 71 37100 54 30 100 72 38100 55 30 100 51 31

Mean 100 53.6 31.7 100 62.8 35.2S.D. - 1.71 2.21 0 7.16 2.78% C.V. - 3.2 7.0 0 11.4 7.9

Standard Spinach

In a separate experiment, both spiked spinach extract and a solvent standard wereinjected 10 times, and mass spectra were obtained under CID conditions.

This table compares the methomyl ion ratios for the two samples. Ion ratios arenormalized relative to the most intense ion at m/z 88.

There is good agreement between the two data sets, indicating the utility of CIDfragmentation for compound confirmation.

Note: More alert people may comment on the fact that the ion ratios expressed inthis table differ from those in the previous slide, in which the ion at m/z 106 was themost intense ion. We believe that this is due to the fact that the data from bothslides was acquired under slightly different conditions and/or on different days.While the table shows good INTR-assay reproducibility, whish is perfectlyacceptable for confirmation of target analytes, some people might be tempted toconclude that CID spectra are well-suited to library matching. This is anassumption that other manufacturers are willing to foster. However, the utility ofCID spectra for library-based identification requires excellent INTER-assayreproducibility, which front-end CID does not offer. This is a very importantdistinction which should be borne in mind when addressing claims of othermanufacturers.

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© 2000 Waters Corp

0

1 0

20

3 0

40

5 0

60

7 0

80

90

1 00

Re

lati

ve A

bu

nd

an

c

S tandard S pinach

m/z 88 m/z 106 m/z 163

Methomyl Confirmation in Spinach

This is a more graphic representation of the results presented in the previous table.It is clear that compound identity can be confirmed even in the presence of acomplex matrix.

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© 2000 Waters Corp

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00

Time

0

100

%

7.04e514.69

6.45

2.271.66

10.277.69

15.53

19.29

Carbaryl, monitoringm/z 145, 202

Propoxur,monitoring m/z 210

Methomyl, monitoringm/z 88, 106, 122, 163

Composite of a Three Component StandardSIR of 7 Channels

In a separate experiment, the chromatogram of three carbamates; methomyl,propoxur, and carbaryl, is displayed. They were analyzed by monitoring the sevenfragment ions corresponding to the three compounds.

83

© 2000 Waters Corp

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340m/z

0

100

%

0

100

%

0

100

%

SIR of 7 Channels ES+ 1.02e5

145

88

202

163 210


210

88 122106 202


106

88

163122 145 210

Carbaryl response at 15.53 min.

Propoxur response at 14.69 min.

Methomyl response at 6.45 min.

Characteristic Mass Spectral Behavior

Here is the corresponding mass spectra obtained from the three carbamates.

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© 2000 Waters Corp

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00

Time

0

100

%

SIR acquisition, extracted chromatogram at m/z 162.9

10.13

9.78

2.19

0.723.97

Peak at 6.42 min from Methomyl

25 pg on-column

S/N ~ 10:1

Detection of Methomyl in Orange JuiceExtract, 8.5 ng/ml, 3 µµµµl Injection

An orange juice extract was analyzed under the same conditions. Shown here is theextracted chromatogram at m/z 162.9 from the orange juice sample detectingmethomyl at 8.5 ng/ml (25 pg on column) with excellent signal/noise ratio.

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© 2000 Waters Corp

Conclusion

• LC/MS adds an additional dimension to on-linechromatographic analyses.– “Resolution” of co-eluting peaks

– Detection of weak chromophores

• LC/MS enables you to perform qualitative and/orquantitative analyses.– SIR for sensitivity

– CID for structural information

It should be pointed out that all of the preceding data was derived from a variety ofexperiments performed over several months in different laboratories. Theyrepresent ‘real’ results; no effort was made to produce “perfect” data. What isimportant is that, taken together, they all tell the same story, lab-to-lab, operator-to-operator, and day-to-day. Hopefully, the moral is clear:

First, LC/MS adds a third dimension to typical on-line separations. It allows you to“peek” under a chromatographic peak. As we saw, this allows the chemist a lot ofleeway when it comes to methods development. As was shown, the massspectrometer can ‘resolve’ co-eluting peaks, allowing significant reductions in runtime. Response does not require a chromophoric or fluorescent group, so there isnot need for derivatization, again, simplifying the analysis.

Second, LC/MS is unique in that it can provide very sensitive and specificquantitative information (when operated in the SIR mode), as well as qualitative,structural information (full-scan analysis provides molecular weight, CID providesstructurally-significant fragment ions). Other analytical instruments are limited toone or the other.

86

© 2000 Waters Corp

Polymer Additive Analysis byEI and APCI

Kate Yu, Eric Block

LC/MS

Waters Corporation

Introduction to polymer additive analysis by LC/MS.

87

© 2000 Waters Corp

Electron Ionization (EI) Advantages

• Classical spectra

• Library searchable

• Positive compound ID

• Relatively easy to obtain

• Rugged

• Interpretable

• Structural elucidation

The Thermabeam interface, which is an improved form of particle beam interface,is used to generate electron ionization (EI) spectra for LC/MS. Electron ionization,due to it’s high ionization energy (about 70 eV), offers a reproducible fragmentationpattern of a molecule, which can be searched against a library for positivecompound identification. This type of ionization is well suited for qualitativeanalysis where extensive structure-informative fragmentation is highly desirable.

88

© 2000 Waters Corp

Advantages of Atmospheric PressureChemical Ionization (APCI)

• Molecular weight information

• Fragmentation with CID

• Easy to use

• Rugged Technique

• Accommodate LC flows up to 2.0 ml/min.

• Good sensitivity

The atmospheric pressure ionization interface is now one of the most popularinterfaces for the single quadrupole LC/MS. In this type of interface, two types ofionization are possible: Electrospray (ESI) and atmospheric pressure chemicalionization (APCI). Both ESI and APCI are soft ionization techniques, which allowmolecular weight information to be obtained easily. In addition, they are both muchmore sensitive and very well suited for quantitative analysis.

89

© 2000 Waters Corp

Choice of EI and APCI

Unknown Compound ID Known Confirmation

MS Detection

Samples,Info

Needed,Lab

Priorities

Waters ZMD(APCI / ESI)

Waters TMD EI

PD

A

The proper choice of interface depends on the information required from theanalysis. If you need to identify unknowns in your sample then EI is the properchoice. For molecular weight information and confirmation of known compounds,either APCI or ESI is best suited.

90

© 2000 Waters Corp

Goal

• To demonstrate how EI and APCI cancomplement each other in chemical analyses

– AcCN extract of Polypropylene as modelsystem

– EI for positive ID

– APCI for confirmation

– Sensitivity and linearity evaluation of EIand APCI

In this work, we chose the organic extractable from polypropylene as our modelanalyte to demonstrate how information separately obtained from EI and APCI cancompliment each other.

91

© 2000 Waters Corp

EI Experimental Conditions

• Integrity MS Detector:

– Scan Range: 60 to 900 Da

– Ion Source Temp: 200oC

– Nebulizer Temp: 75oC

– Expansion Region Temp: 75oC

In our lab, the polypropylene was extracted with acetonitrile. 10mL of acetonitrilewas added to 5 grams of polypropylene and heated to 60°C for 72 hours. Thesupernatant was filtered prior to analysis. The acetonitrile extracts were thenanalyzed by two LC/MS instruments: First, the Thermabeam interface with EIoffered on the Waters Integrity System. Shown here are the instrument conditionsfor the Integrity mass detector.

92

© 2000 Waters Corp

APCI Experimental Conditions

• Platform LC Conditions:

– Scan Range: 80 to 1000 Da

– Ionization Type: APCI Negative

– Cone Voltage: -12 V (Regular)

– Cone Voltage: -40 V (CID)

– Probe Temperature: 400oC

Second, the Atmospheric pressure Ionization Interface with APCI (or ESI) offeredon the Micromass Platform LC mass detector. Shown here are the instrumentconditions for the Platform LC mass detector. PDA detection was available on bothinstruments.

93

© 2000 Waters Corp

LC/MS of AcCN Extract from PP

• Total ion chromatograms (TIC) of the extract:

– Comparison of EI, APCI-, and PDA

• EI analysis of each component:

– Spectrum of the unknown

– Chemical structure

• APCI analysis of each component:

– Spectrum of the unknown with MW information

– CID Spectrum of the unknown with limitedfragmentation

Compound identification was determined by;

1. Comparison of the three modes of detection.

2. Matching EI spectra with the Wiley library generates lead suspects.

3. Obtaining molecular weight information from APCI helps rule out wrongmatches.

4. Generating limited fragmentation in API with In-Source Collision InducedDissociation (CID) helps to confirm EI results.

94

© 2000 Waters Corp

APCI-, EI, PDA Comparison

Min

EI: TIC

12 14 16 18 20 22 24 26 28 30

PDA: 210 nm

3

2

754

1

1

1

2

2

4

4

3

3

5

5

6

7

7

APCI-: TIC

Shown here is the comparison of three modes of detection of the polypropyleneextract.

95

© 2000 Waters Corp

MS Spectra of Peak #1

m/z100 200 300 400 500

48.4

67.9

127.0

149.0159.1

219.1

232.1

279.3 365.1436.0

200 300 400 500 600 700 800m/z

435

433

431

436

437

[M-H]--

APCI- : 12V

200 300 400 500 600 700 800m/z

277

231257

278 433279 431 436

APCI-: 40V

EI

HO

t-BuO

O

t-Bu

NHO

NH

O

OH

MW 436

Note that two mass spec interfaces generate different mass spectra.

The spectra on the left, comes from the APCI interface. The upper left corner massspectrum was created using a cone voltage of 12 volts.The major intensity indicatesthe molecular ion (435 [M-H]-). Increasing the cone voltage, creating In-SourceCID, generates limited fragmentation displayed in the lower left corner.

The mass spectrum in the upper right corner generated by the EI interface createslibrary searchable spectra. Information about the chemical structure can beobtained.

96

© 2000 Waters Corp

MS Spectra of peak #656.6

69.082.9

97.0

111.0

125.1139.1

153.1168.1

182.1196.2

210.2224.2

252.2

60.00 100.00 140.00 180.00 220.00

57.0

m/z

43.069.0

83.0 97.0

111.0

125.0

139.0 153.0168.0

182.0196.0

210.0224.0 252.0

EI: Unknown Spectrum

EI: Library spectrum, 96% Match

H3C (CH

2)15

CH CH 2

Positive identification of this unknown compound was determined by comparingthe EI mass spectrum of peak # 6 (upper spectrum) to the Wiley library and comingup with an excellent match (lower spectrum), 1-Octodecanol.

97

© 2000 Waters Corp

AcCN Extract of PP (Identification)

• 1. Naugard Degradant: MW 436

• 2. NC-4: MW 414

• 3. 2-Bis(methylthio)methylene-1-phenyl-1,4-methyl-4-penten-1-one: MW 278

• 4. 7,9-Di-tert-butyl-1-oxaspiro[4,5]deca-6,9-diene-2,8-dione: MW 276

• 5. Naugard: MW 696

• 6. 1-Octodecene: MW 252

• 7. Irgonox 1076: MW 530

10 12 14 16 18 20 22 24 26 28 30Minutes

12

34

7

6

5

EI: TIC

Utilizing the both EI and APCI spectra all of the compounds detected in thepolypropylene extract were identified.

98

© 2000 Waters Corp

Results of a 20-Fold Dilution

Minutes

10 12 14 16 18 20 22 24 26 28 30

3

2

54

7

6

TIC of APCI-

TIC of EI

PDA at 210 nm

A 20-fold dilution of the polypropylene extract was analyzed by the three detectionmodes. The APCI interface demonstrated the best sensitivity.

99

© 2000 Waters Corp

Conclusions

• Major peaks identified by EI and confirmed byAPCI-

• Comparable chromatograms by PDA and MS

• Hydrocarbon component can ONLY bedetected by EI

• APCI- exhibited better sensitivity and linearitythan EI

Among various analytical techniques used by people for additive analysis, LC/MSis superior. In addition to the chromatographic separation obtained with HPLC, amass spectrometer offers sensitive and quantitative detection and positiveidentification of the additives.

1

© 2000 Waters Corp

Natural Products Analysis

!Food Supplements, Botanical Extracts• St.Johns Wort, and Red Clover

!Strong Marketing Claims• Limited Substantiation

!Is the Marketed Compound the Active Ingredient?• Questionable

!Regulation by FDA Restricted by Statute• Dietary Supplement & Health Education Act (DSHEA)

1994

The identification and quantitation of compounds extracted from natural productsare method development challenges. Today we will discuss analytical methodsdeveloped for the analysis of St. John’s Wort plant and red clover.

The nutraceutical industry is growing rapidly. There are a number of naturalproducts on the market today proclaiming health benefits with limited or no clinicaltrials.

Scientists are questioning what is the real active ingredient in some of these naturalproducts.

Due to DSHEA, the Food and Drug Administration (FDA) has less oversight overnutraceuticals than pharmaceuticals.

2

© 2000 Waters Corp

Natural Products AnalysisQuality Concerns

!Quality of Botanicals varies by species, location,season, and “Mother Nature”

!Identification and Characterization of thebotanical species is subjective

!Few Official Methods of analysis are available

!Liquid Chromatography Methods utilizingPhoto Diode Array and Mass Spectrometry detection are being developed

Since the raw material of these natural products come from worldwide sourcesgrowing conditions differ creating variablitiy in the final product.

The matrix of these botanicals are complex, making identification andcharacterization of the species difficult.

Few validated analytical methods currently exist for nutraceuticals.

However, analytical methods utilizing HPLC with PDA and MS are beingdeveloped for these natural products.

3

© 2000 Waters Corp

Natural Products AnalysisAnalytical Needs

! Manufacturers use specific marker compounds for Quality Assurance

• Hypericin in St. Johns Wort• Isoflavonoids in Red Clover

! Method Validation for several components being conducted by:

• Institute for Nutraceutical Advancement• American Society of Pharmacognosy

Currently nutraceutical use marker compounds for quality assurance such ashypericin in St. Johns Wort and isoflavonoids in red clover.

Citing the need for consistency and consumer confidence in the market place,manufactures are moving to more science and validated methods from consensusorganizations.

The Institute for Nutraceutical Advancement, sponsored by 30 companies, has aMethod Validation Program developing HPLC-based methods.

The American Society of Pharmacognosy is another organization developingmethods for nutraceuticals.

4

© 2000 Waters Corp

Hypericin Analysis by LC/MS

Jeanne B. Li and Michael P. Balogh

LC/MS

Waters Corporation

LC•GC , Vol 17, No. 6, June 1999

Introduction to hypericin analysis by LC/MS

5

© 2000 Waters Corp

Hypericin and Related Compounds

CH 3

OH O OH

CH 3

HO

HO

OH OHO

HypericinMW 504.45

PseudohypericinMW 520.45

OH O OH

CH2

CH3

HO

HO

OH OHO

OH

OH O OH

CH3

CH3

HO

HO

OH OHO

ProtohypericinMW 506.45

ProtopseudohypericinMW 522.45

OH O OH

CH 3

CH3

HO

HO

OH OHO

OH

* *

The structures of the compounds of interest in the plant extract are shown in thisslide. Note the closing of the ring E on the right-hand side of hypericin andpseudohypericin. This makes them more hydrophobic which effects their elutionorder in reversed-phase separations.

The protohypericin and protopseudohypericin are the biosynthetic precursors thatare easily converted to hypericin and psuedohypericin.

6

© 2000 Waters Corp

Hypericin AnalysisChromatographic Conditions

Column: Waters Symmetry C8, 2.1 mm x 150 mm

Mobile Phase: A = 100mM TEA•Acetate, pH 7 B = Methanol

C = Acetonitrile

Gradient: Linear over 15 minutes 30:39:31 to 10:50:40


Detection: PDA at 588 nm

Mass Spec: Negative Electrospray Ionization (ESI-)

Here are the optimized chromatoghraphic conditions for hypericin analysis. BothPDA and MS detectors were used.

7

© 2000 Waters Corp

Hypericin UV/Vis SpectraTEA-Acetate vs. TriFluoroAcetate (TFA)

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

AU

nm250 300 350 400 450 500 550 600 650 700

588

TEA-Acetate

TEA-TFA

The absorbance spectra of hypericin in trifluoroacetic acid and intriethylammonium acetate are shown in this slide. The unique UV-VIS spectrum ofnaphthodianthrones has much stronger absorbance at 588 nm in triethylamineacetate than in trifluoroactic acid. This buffer also was conducive to goodionization by electrospray of hypericin and the other compounds in negative mode.

8

© 2000 Waters Corp

Hypericin SpectraPDA and ESI--MS

600250 300 350 400 450 500 550 650nm

588285

328547

250 300 350 400 450 500 550 600 650

m/z

503

504

505

Abso

rban

ceIn

tensi

ty [M-H] -

PDA Spectrum

Mass Spectrum

A comparison of the UV-VIS spectrum and mass spectrum of the hypericin peak.

9

© 2000 Waters Corp

Hypericin Standard ChromatogramsPhoto Diode Array and ESI--MS

Abso

rbance

Inte

nsi

ty

588 nmλ Max

m/z 503[M-H]-

Minutes

2 4 6 8 10 12 140

Extracted chromatograms from the separation of hypericin standard monitoredsimultaneously by PDA detection at 588 nm and MS detection at m/z 503.

10

© 2000 Waters Corp

Plant Extract ChromatogramsPDA & ESI-

1.20

4.79

10.67

588 nm

12.18

11.22 12.84

T IC

2 4 6 8 10 12 14

Minutes0

Abso

rbance

Inte

nsi

ty

Hypericin

The chromatograms of the plant extract of sample 1 obtained by monitoringaborbance at 588 nm (upper chromatogram) and the total ion current (lowerchromatogram). The peak at 10.67 minutes is hypericin.

11

© 2000 Waters Corp

Plant Extract #1 - Extracted Masses[M-H]-

9.48

6.841 0.86

m/z 503

m/z 521

m/z 505

m/z 519

588 nm

Inte

nsity

AU

1 0.74

Minutes

142 4 6 8 10 120

1.204.79

1 0.67

4.863.12

4.26

11.52Protopseudohypericin

Pseudohypericin

Hypericin

Shown here are the extracted mass chromatograms of the plant extract from sample1 with the UV absorbance chromatogram on the bottom. Note that protohypericinwas not detected in this sample.

12

© 2000 Waters Corp

ESI- Mass Spectra - Plant Extract #1

521341326

315

139

583567522 584

519275

385523

543

551

279 383353

553

555585

100 150 200 250 300 350 400 450 500 550 600

m/z

503417

347349 504

P ea k4.26 min

4.86 min

9.48 min

10.74 min

Inte

nsity

Protopseudohypericin

Pseudohypericin

Hypericin

Shown in this slide are the mass spectra extracted from the chromatographic peaksin sample 1.

13

© 2000 Waters Corp

Plant Extract #2 - Extracted Masses[M-H]-

m/z 503

m/z 521

m/z 505

m/z 519

588 nm

Inte

nsity

AU

M inutes

142 4 6 8 10 120

4.26

11.70

4.80

9.72

10.92

10.92

4.721.17 10.82


Pseudohypericin

Protohypericin

Hypericin

Shown here are the extracted mass chromatograms of the plant extract from sample2 with the UV absorbance chromatogram on the bottom.

14

© 2000 Waters Corp

ESI- Mass Spectra - Plant Extract #2521

522

51 9

5 20

5 515052 79

1 79 4 955 06

5 53

5 54

100 150 200 250 300 350 400 450 500 550 600

m /z

503

119 4 715 04

Pe ak4.26 m in

4.80 m in

9.72 m in

10.92 m in

Inte

nsi

ty


Pseudohypericin

Protohypericin

Hypericin

Shown in this slide are the mass spectra extracted from the chromatographic peaksin sample 2.

15

© 2000 Waters Corp

Comparison of Plant Extracts

m /z 503

m /z 521

m /z 505

m /z 51 9

588 nm

9 .4 8

6 .8 4

10 .8 6

Inte

nsity

AU

10 .7 4

Mi nutes

142 4 6 8 10 120

1 .2 0

4 .7 910 .6 7

4 .8 63 .1 2

4 .2 6

11 .5 2

Mi nutes

142 4 6 8 10 120

4. 26

1 1. 70

4. 80

9. 72

1 0. 92

1 0. 92

4. 72

1. 17 1 0. 82

Plant Extract #2Plant Extract #1

Comparison of plant extracts 1 and 2 indicate that more of the hypericin compoundswere solubilized from the plant powder of sample 2. The hypericin content in plantsreportedly changes as a function of climate and season.

16

© 2000 Waters Corp

In Source Collision-InducedDissociation of Hypericin

Low cone voltage

High cone voltage

503

405

433431406 487459

458444434

504

m/z340 360 380 400 420 440 460 480 500 520

503

504

Inte

nsity

In-source Collision Induced Dissociation (CID) of hypericin in a single-quadrupolemass detector. Shown are the spectra at high (upper spectrum) and low (lowerspectrum) cone voltages. The low cone voltage was optimized for sensitivity of the[M-H]- ion at M/Z 503.

17

© 2000 Waters Corp

Natural Product AnalysisPhytoestrogens-Isoflavonoids

!Electrospray, is a soft ionization, yielding a single[M+H]+ or [M-H]- line

!Gives some fragmentation for structuralidentification, but no Libraries available to search

!Electron Ionization, Particle Beam, yields information rich spectra

!Can deduce molecular structure and libraries areavailable for Unknown Identification

A group of isoflavonoids commonly called phytoestrogens are found in red clover.

Analysis of plant extracts by LC/MS using an electrospray interface, which is a softionization technique, yields a single molecular ion with limited fragmentation.

The Thermabeam interface, which is an improved form of particle beam interface,is used to generate electron ionization (EI) spectra for LC/MS. Electron ionization,due to it’s high ionization energy (about 70 eV), offers a reproducible fragmentationpattern of a molecule, which can be searched against a library for positivecompound identification. This type of ionization is well suited for qualitativeanalysis where extensive structure-informative fragmentation is highly desirable.

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© 2000 Waters Corp

Characterization of Plant ExtractIsoflavonoids Using LC-PDA-Mass Spec

Michael P. Balogh

LC/MSWaters Corporation

Published inLC•GC, Vol 15, No. 5, May 1997

Introduction slide to the analysis of isoflavonoids in red clover using the WatersIntegrity LC/MS System.

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© 2000 Waters Corp

Natural Product AnalysisRed Clover Isoflavonoid Characterization

Chromatographic Approach

Column: Waters Symmetry C8, 3mm x 150mmMobile Phase: AcCN / Water Linear Gradient

15/85 to 36/64 over 40 minutesFlow Rate: 400 µL/minDetector 1: PDA Scan from 200 to 600 nmDetector 2: Mass Spec, Electron Ionization Scan from50 to 500 m/z

Here are the optimized chromatographic conditions for the analysis. Both PDA andMS detection are employed.

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© 2000 Waters Corp

Natural Product AnalysisRed Clover Isoflavonid Characterization

Minutes

* Found in Wiley Library via AutoSearch

MS Total Ion Current Chromatogram

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00

PDA MaxPlot Chromatogram

1*

2*

3* 5*6* 7

8*

9*

10*

11*13*

14*

4*

12*

1 Inositol 2 Ononin (7-Hydroxy-4’-methoxyisoflavone-7-O-Glucoside) 3 Daidzein (7,4’-DihdroxyIsoflavone) 4 Sissotrin (5,7-Dihydroxy-4’-MethylIsoflavone) 5 Isomer of Peak 4 6 2’,4,7-TriHydroxyIsoflavone) 7 Diometin (3’,5,7-HydroxyFlavone) 8 Formonoetin (7-Hydroxy-4’-MethoxyIsoflavone) 9 Medicarpin10 Biochanin A (5,7-Dihydroxy-4’-MethoxyIsoflavone)11 Phthalate12 Propylene Glycol, Benzoates131,2-BenzeneDiCarboxylic Acid14 Ethyl Linoleolate

Trifolium Pratense(Red Clover)

The chromatographic separation of red clover extract. The PDA plot (lowerchromatogram) and mass spec TIC (upper chromatogram) is shown here. Note thatall of the starred compounds were identified using the commercially availableWiley Library.

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© 2000 Waters Corp

Some Common Flavonoids -their derivatives and secondary metabolites

Medicarpin

OHO

O

OMe

Diosmetin

Biochanin A

OHO

OOHOMe

Genistein

OHO

OOH

OH

Flavones IsoflavonesOMe

OH

HO

O

O

Acacetin

OMe

OHO

OOH

OH

Shown here are some of the structures of the flavones and isoflavones examined inthis study

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© 2000 Waters Corp


Minutes

MS Total Ion CurrentChromatogram BA

C

35.00

1 2

34

PDA UV Chromatogram

15.00 20.00 25.00 30.00

1 Ononin (7-Hydroxy-4’-methoxyisoflavone-7-O-Glucoside) A Daidzein (7,4’-DihdroxyIsoflavone) 2 Sissotrin (5,7-Dihydroxy-4’-Methylsoflavone) B Diosmetin (3’,5,7-HydroxyFlavone) or Pratensein (3’,5,7-Trihydroxy-4’-MethoxyIsoflavone) 3 Formononetin (7-Hydroxy-4’-MethoxyFlavone C Medicarpin (3-Hydroxy-9-Methoxyptererocarpen) 4 Biochanin A (5,7-Dihydroxy-4’-MethoxyIsoflavone)

Identification of daidzein, diodmetin, and medicarpin in the red clover extract fromthe west coast.

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© 2000 Waters Corp


Identification of Close-Eluting Peaks by MS

HO O

O OH

(M+)254

Daidzein(4’,7-DiHydroxyIsoflavone)C15H10O4 MW = 254.23

237225

197168

137

127

118

10889

80

PDA RT = 15.58MS RT = 16.95

A

20.0015.00

(M + -162)

268

PDA RT = 14.91MS RT = 15.64

Glu-O O

O OCH3

(M+)430310

296

284

253

240225

197168

132

1088960

Ononin(7-Hydroxy-4’-MethoxyIspflavone-7-O-Glucoside)

C22H22O9 MW = 430.46

A close up view of the separation shows a very small peak (A) identified as theaglycone daidzein at the trailing edge of the glycoside ononin. The mass spectra foreach are significantly different in this case, which allows direct identification bylibrary match from a single experiment. The molecular ion of the large peak(ononin) at m/z 430 is followed by the loss of glucose (M+-162) forming the basepeak of the spectrum. The smaller peak (daidzein) produces the molecular ion atm/z 254 as the base peak of the spectrum.

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© 2000 Waters Corp

Natural Product AnalysisRed Clover Isoflavonid Characterization

PDA Differentiation of Isomers

OHO

OOH

OH

OMe

m/z

69

OHO

O

OH

OHOMe

69

77 95105

133153

174 213

229257

271285

300

m/z

Probable Pratensein

380240 260 280 300 320 340 360nm

380240 260 280 300 320 340 360nm

Diosmetin Standard

Acquired Peak

B

25.0020.00

262.3 nm

344.5 nm

77 105120 134

153

167178

229

239

257

271

285

300

Comparison of the acquired peak with the UV spectrum of diosmetin standard,indicates that the peak is probably pratensein. Both diosmetin and pratensein havea formula weight of 300.27.

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© 2000 Waters Corp

Natural Product and NutraceuticalFutures

!Increased use of Mass Spectrometry to complimentPhoto Diode Array detection for Product andComponent characterization

!Need for Validated Analytical Methods for ProductTarget Compounds

!Need for Standardization among Suppliers andManufacturers for product consistency

!More FDA Regulation in the Future?

Waters foresees increased use of HPLC and LC/MS for the analysis ofnutraceuticals.

More validated analytical methods are required.

Suppliers and manufacturers need to follow the same quality assurance proceduresto assure product consistency.

Will the FDA regulate the nutraceutical industry more closely in the future?

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© 2000 Waters Corp

Successful Analyses

Sugars C18 Sep-Pak Carbohydrate HPLC/RI

EED’s Oasis HLB SymmetryShieldRP18 & RP8

HPLC/PDALC/MS

PAH’s Oasis HLB LiChrospherePAH

HPLC/PDA& Fluorescence

Carbamates Oasis HLB Carbamate HPLC/PCFDLC/MS

Pesticides Oasis HLB Symmetry C18 LC/MS

PolymerAdditives Org. Extract Symmetry C18 LC/MS

Nutraceuticals Org. Extract Symmetry C8 LC/MS

Application Sample Prep Column Technology

In summary, this seminar has demonstrated successful analyses for a number ofapplications utilizing a variety of Waters products and technologies.

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© 2000 Waters Corp

Agricultural ChemicalsResidues in Food & Water

Fine ChemicalsPesticidesPolymer Additives

Functional Foods & Dietary SupplementsNutrientsNutraceuticals

Environmental ContaminantsEndocrine Disruptors

Conclusion: Waters offers solutions to a wide range of analytical challenges in theindustrial market place.

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© 2000 Waters Corp

Ι Bisphenol A & Nonylphenol

ΙΙ Pesticides

Nihon Waters

LC/MS Application Laboratory

Jun Yonekubo

Developing LC/MS Methods for:

Introduction to analytical methods developed in Japan. First, a method for theanalysis of Bisphenol A and nonylphenol using LC/MS.

Second, an LC/MS method for the analysis of some new pesticides in Japan.

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© 2000 Waters Corp

Chromatographic Conditions

HPLC: Waters Alliance® SystemColumn: Waters Symmetry® C8 , 2.1 x150 mmTemperature: 40° CInjection Volume: 10µLMobile Phase:

A: 100% DI WaterB: 100% Acetonitrile


Gradient: Time %A %B Curve Initial 60 405 25 75 6

11 60 40 11

This gradient/detection method is improved over a previous method for

Bisphenol A. This new method provides the capability of shorter gradient methodwith C8 50mm column. Run time (include equilibration) is reduced from 25 to 17min.(8 min.)

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© 2000 Waters Corp

Mass Spectrometer Conditions

Instrument: Waters ZMD Zspray ™ Mass Detector

Interface: Negative Electrospray (ESI-)

Scan Function: Multiple Selected-Ion Recording (SIR):

Time Cone Dwell Group mins. Compound Mass Voltage Time

1 0-5 Bisphenol A 226.9 39V 1.0 secs

2 5-11 Nonylphenol 219.0 39V 1.0 secs

Software MassLynx ™

This method also incorporates a reduction of Multiplier V from ca.900 to 650 asnormal voltage.

Monitoring ion m/z=227 and 219 are each compound’s pseudomolecular ion (M-H)- on ESI negative mode.

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© 2000 Waters Corp

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

Time

-4

100

%

2.05

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

Time

-2

100

%

2.03

Bisphenol A

n=5 Overlay Chromatogram%RSD (area ) = 2.64Std Dev (conc.) = 0.03

S/N=3.1

SIR Chromatograms of 0.5ppb Bisphenol A

This slide show 5 SIR mass chromatograms of 0.5ppb Bisphenol A std.(S/N>3)

% RSD calculated from this data is 2.64 (Area) and Standard Deviation is 0.03(Concentration)

F.Y.I.

HP domestic toss-sheet value 50ppb %RSD 2.13

I reported S/N value as RMS previously, but PtP algorithm is more familiar inJapan.

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© 2000 Waters Corp

6.00 7.00 8.00 9.00 10.00 11.00 12.00

Time

44

100

%

7.57

6.00 7.00 8.00 9.00 10.00 11.00 12.00Time

39

100

%

7.56

SIR Chromatograms of 5ppb Nonylphenol

n=5 Overlay Chromatogram%RSD (area) = 3.16Std Dev (conc.) = 0.24

S/N=18.0

This slide show 5 SIR mass chromatograms of 5ppb Nonylphenol. (S/N>18)

% RSD calculated from this data is 3.16 (Area) and Standard Deviation is 0.24(Concentration).

F.Y.I.

HP domestic toss-sheet value 50ppb %RSD 2.34

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© 2000 Waters Corp

Bisphenol A and Nonylphenol in BlankSolution from SPE (PS-2) Procedure

Conditioning5mL ACN/ 5mL DIW

Loadup to 1000mL DIW

Elution1mL ACN 1mL CH2Cl2

Evaporate Reconstitute

1mL ACN

Blank tapBlank tap Blank KNGBlank KNG

This slide show the sample preparation procedure of two different sources ofdeionized water, both obtained from Millipore water purification systems. TheBlank Tap sample came from the Tokyo Water Institute and the Blank KNG samplecame from the Kanagawa Health Institute.

Both blanks represent a 1000 fold sample enrichment using a PS-2 solid phaseextraction cartridge.

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© 2000 Waters Corp

2.00 4.00 6.00 8.00 10.00 12.00

Time

5

100

%

White: Blank Tap - Tokyo Institute DI WaterBisphenol A (0 ppb) Green: Blank KNG - Kanagawa Institute DI WaterBisphenol A (2.10 ppb)

Blue: Blank Tap - Tokyo Institute DI WaterNonylphenol (30.75 ppb)Red: Blank KNG - Kanagawa Institute DI WaterNonylphenol (3.70 ppb)

SIR Chromatograms of Blanks

This slide shows an overlay of the SIR mass chromatograms of the two Blanks.

Note that Bisphenol A and Nonylphenol were both detected in the Blank KNG.

Only Nonylphenol was detected in the Blank Tap.

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© 2000 Waters Corp

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0

ppb

0

1.57e4

Response

Blank KNG Analyte 2.10 ppb

Bisphenol A Calibration Curve; 0.1-10 ppb

Compound 1 name: Bisphenol A (m/z 227)Coefficient of Determination: 0.996236Response type: External Std, AreaCurve type: Linear, Weighting: Null

The method displays good linearity for Bisphenol A.

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© 2000 Waters Corp

0.0 20.0 40.0 60.0 80.0 100.0ppb

0

4.76e4

ResponseBlank tap Analyte 30.75 ppb

Blank_KNG Analyte 3.70 ppb

Nonylphenol Calibration Curve; 1-100 ppb

Compound 2 name: Nonylphenol (m/z 219)Coefficient of Determination: 0.999657Response type: External Std, AreaCurve type: Linear, Weighting: Null

Nonylphenol also demonstrated excellent linearity.

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© 2000 Waters Corp

1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00

Time

12

100

%2.12

Blank

Sample

1.83ppb Bis-phenol A

Detection of Bisphenol A inPolycarbonate Extract

This slide shows the actual sample (Hot water extracts of polycarbonate tableware)analysis.

Polycarbonate materials are used to manufacture utensils in Japanese school lunchknown as "Kyuusyoku".

This sample is an example of Bisphenol A leaching out from a hot water washingprocess.

Concentration of Bisphenol A from this sample (1.83ppb) is estimated.

This concentration takes into account the blank.

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© 2000 Waters Corp

Chromatographic Conditions

HPLC: Waters Alliance® SystemColumn: Waters Symmetry® C18, 2.1 x150 mmTemperature: 40° CInjection Volume: 10µLMobile Phase:

A: 0.1% Acetic AcidB:100% Acetonitrile


Gradient: Time %A %B Curve Initial 65 3530 20 80 645 65 35 11

This gradient/detection method was developed for the analysis of nine pesticidesthat are used in Japan.

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© 2000 Waters Corp

Mass Spectrometer Conditions

Instrument: Waters ZMD Zspray ™ Mass Detector

Interface: Electrospray (ESI)

Scan Function:

MassLynx ™ Software

Scan ESI+ and ESI- simultaneously

Mass Range: 130-600

Cone Voltage: 35

The electrospray interface for the ZMD was employed for this application, runningin positive and negative mode simultaneously.

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© 2000 Waters Corp

Compounds Conc.(ppm)1.Diflubenzuron 2.562.Tebufenozide 4.923.Iprodione met. 5.244.Flusulfamide 5.245.Phoxim 2.486.Fenpyroximate 2.507.Flufenoxuron 3.008.Chlorfluazuron 5.289.Methoprene 2.52

10.00 14.00 18.00 22.00 26.00 30.00 34.00Time12

100

%

3

100

%

-3

100

%9

8

76

54

3

21

UV 254 & 270 nm

ESI positive

ESI negative

Separation and Detection of 9 Pesticides

The Waters 996 PDA detector was also utilized for the method. Note that in theupper chromatogram peaks 3 and 4 are not baseline resolved using UV detection.By combining the ESI positive and negative TIC chromatograms all nine peaks areseparated and detected.

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© 2000 Waters Corp

Standard

19.00 20.00 21.00 22.00 23.00Time

0

100

%

0

100

%

-2

100

%

PDA 254 nm

Scan ES+

Scan ES-

54

3 Citrus spiked,Symmetry C18

19.00 20.00 21.00 22.00 23.00Time

0

100

%

27

100

%

-556

100

%

PDA 254 nm

SIR of 3 Channels ES+

SIR of 2 Channels ES-

5?

3+4

3.Iprodione met. 5.24ppm4.Flusulfamide 5.24ppm5.Phoxim 2.48ppm

Selectivity of MS Detection

3.Iprodione met. 52.4ppb4.Flusulfamide 52.4ppb5.Phoxim 24.8ppb

Comparison of a standard to a spiked grapefruit extract demonstrates the selectivityof MS detection for this application.

Note in the upper chromatograms that peaks 3 & 4 coelute and peak 5 is notdetected by the PDA detector.

Utilizing the electrospray interface, the spiked pesticides in the sample are detectedand quantitated at the part per billion level (ppb).

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© 2000 Waters Corp

200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500m/z

0

100

%

0

100

%

0

100

%

0

100

%

1: Scan ES+ 330.0

221.0 288.0244.0 316.3 371.0353.2

2: Scan ES- 412.9

258.91: Scan ES+

257.0215.9

271.0 299.1285.0

435.01: Scan ES+

366.1

216.1 422.2

Fenpyroximate, 24.40 min

Phoxim, 21.56 min

Flusulfamide, 20.64 min

Iprodione met., 20.35 min

ESI Mass Spectra - 4 Pesticides

The mass spectra of four of the pesticides extracted from the standard TICchromatogram is displayed here.

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© 2000 Waters Corp

15.00 20.00 25.00 30.00 35.00Time

16

100%

2

100%

1

100

%

0

100%

31

100%

0

100%

41

100%

3: SIR of 1 Channel ES+

m/z 311.00

4: SIR of 2 Channels ES-

m/z 351.00+413.00

5: SIR of 3 Channels ES+

m/z 330.00+156.00

5: SIR of 3 Channels ES+ m/z 366.00


m/z 489.00

7: SIR of 2 Channels ES- m/z 520.00

8: SIR of 1 Channel ES+ m/z 191.00

5.Phoxim

4.Flusulfamide

9.Methoprene

8.Chlorfluazuron

7.Flufenoxuron

6.Fenpyroximate

3.Iprodione met.

2.Tebufenozide

1.DiflubenzuronCompounds Conc.(ppb)1.Diflubenzuron 25.62.Tebufenozide 49.23.Iprodione met. 52.44.Flusulfamide 52.45.Phoxim 24.86.Fenpyroximate 25.07.Flufenoxuron 30.08.Chlorfluazuron 52.89.Methoprene 25.2

Composite SIR Chromatograms of 9 Pesticides

These SIR chromatograms of a 25-52 parts per billion (ppb) standard mixture ofthe nine pesticides demonstrate the excellent sensitivity of the Waters ZMDdetector.

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© 2000 Waters Corp

15.00 20.00 25.00 30.00 35.00

Time

11

100%

0

100%

17

100%

5

100%

27

100%

0

100%

33

100%


m/z 311.00


m/z 351.00+413.00

5: SIR of 3 Channels ES+

m/z 156.00+330.00

5: SIR of 3 Channels ES+ m/z 366.00


m/z 489.00


m/z 520.00

8: SIR of 1 Channel ES+ m/z 191.009.Methoprene

8.Chlorfluazuron

7.Flufenoxuron

6.Fenpyroximate

5.Phoxim3.Iprodione met.

4.Flusulfamide2.Tebufenozide

1.Diflubenzuron

SIR Chromatograms of Spiked Grapefruit

Compounds Conc.(ppb)1.Diflubenzuron 35.14 *2.Tebufenozide 51.593.Iprodione met. 53.334.Flusulfamide 73.66 *5.Phoxim 31.38 *6.Fenpyroximate 23.127.Flufenoxuron 31.028.Chlorfluazuron 49.019.Methoprene 38.05 *

* Detected in non-spiked sample

All nine pesticides were detected and quantitated at the ppb level in the spikedgrapefruit using the the electrospray interface operating in positive and negativemode simultaneously.

Note that four of the pesticides were also detected in a non-spiked grapefruitextract.

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© 2000 Waters Corp

Conclusion

LC/MS Offers:• Specificity

– SIR

– CID

• Linearity

• Flexibility– Optimize source parameters per Analyte

• Cone voltage/ Time Programming

• +/- switching

LC/MS enables you to perform qualitative and/or quantitative analyses.

SIR for sensitivity and CID for structural information.

The ZMD detector demonstrates good linearity.

The MassLynx software provides flexibility in optimizing the cone voltage usingtime programming and/or operating in positive and negative mode simultaneouslyfor each analyte.

Food for Thought - Universidad De...

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