Introduction to the ACQUITY UPLC H-Class Amino Acid ... · r Met Val NVa Ile Leu Phe Trp Minutes...

©2012 Waters Corporation 1

Introduction to theACQUITY UPLC H-Class

Amino Acid Analysis System

AM

Q

NH

3 His

Ser Arg

Gly

Asp G

lu Thr

Ala P

ro

Der

iv P

eak

Cys

Lys

Tyr

Met V

al IleLe

u

Phe

AU

0 . 0 0

0 .0 2

0 .0 4

0 .0 6

0 .0 8

0 .1 0

0 .1 2

0 .1 4

0 .1 6

0 .1 8

0 .2 0

0 .2 2

M in u te s1 .5 0 2 .0 0 2 .5 0 3 .0 0 3 .5 0 4 .0 0 4 .5 0 5 .0 0 5 .5 0 6 .0 0 6 .5 0 7 .0 0 7 .5 0 8 .0 0


Amino Acid Analysis

Required in many disciplines and applications– Components of proteins– Metabolic intermediates– “Bound vs. Free”– Nutritional value of foods and feeds

Difficult analytical problem– Separation

o Wide range of propertieso Slight differences between pairs

– Detectiono No chromophoreo Wide concentration range


Separation of Amino Acids

Cation-exchange– Original method for amino acid analysis

– Large historical data base of uncommon amino acids

– Realistic run times near 30-60min

Reversed-phase HPLC of derivatized amino acids– Best separation selectivity

– Best sensitivity

– Shortest run times


Detection of Separated Amino Acids

Post-column Reaction– Ninhydrin or OPA with ion-exchange most common

– ESI-MS with reversed phase HPLC and no derivatization

– Pulsed Amperometric Detection

Pre-column derivatization– Many reagents used successfully; some commercialized

– Add UV or fluorescence detectability

– Improve chromatography

– Usually faster and more sensitive

– Successful use requires care


Design Considerations

Base new method on AccQ-Tag™ Derivatization– Established chemistry– Well understood control of reaction– Simple manual derivatization of batches

Base new method on ACQUITY UPLC™ H-Class System– Highest resolution– Well-controlled column chemistry– Quality-tested eluents– Established and available instrumentation

Base new method on Empower Software– Fully-compliance ready– Method templates– Custom calculations


Chemistry of AQC Derivatization

N

N

NH

O

O

O

O N

NH

O

NHOH

O

N

NH2

NH2

O

OH

NH

O

OH

N

N

NH

O OHO

NN

NH

NH

O

AQC Reagent

Derivatized Amino Acids

6-Aminoquinolone (AMQ)

1o Amino Acid;t1/2 <<1 s

2o Amino Acid;t1/2 <<1 s

bis-aminoquinoline urea (derivatization peak)

N-HydroxySuccinimide

+ CO2

+

AMQH2O

t1/2 ~ 15 s


Ultra Performance LC Technology

A class of separation science– Based on chromatography columns with very small particles

– Based on instruments designed to take advantage of the small particles

Improves Resolution, Sensitivity, and Speed with no compromises

Suitable for chromatographic applications in general


UPLC® Amino Acid Analysis Application Solution


Amino Acids Analysis Solution ACQUITY and H-Class

ACQUITYN

H3

HyP

roH

is

Asn

Tau

Ser

Gln

Arg

Gly As

p Glu Th

r

Ala

GA

BA

Pro

HyL

ys1

HyL

ys2 AA

BAO

rn

Cys

Lys

Tyr

Met

Val

NV

a Ile Leu

Phe

Trp

Minutes1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

H-Class

AMQ

NH

3H

yPro

His

Asn

Tau Se

rG

lnAr

gG

ly

Asp

Glu

Thr

Ala

GAB

A Pro

HyL

ys1

HyL

ys2

AABA O

rnD

eriv

Pea

kC

ys Lys

Tyr

Met Va

lN

Va Ile Leu

Phe

Trp

Minutes1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00


ACQUITY UPLC H-Class and H-Class Bio Amino Acid Analysis System


Detector Comparison

AM

Q

NH

3H

yPro

His

Asn T

au Ser

Gln A

rgG

ly

Asp

Glu

Th

r Ala

GA

BA

Pro

HyL

ys1

HyL

ys2

AA

BA

Orn

Der

iv P

eak

Cys

Lys

Tyr

Met

Val NV

a

Ile

Leu

Phe Trp

AU

0.00

0.05

0.10

Minutes0.00 2.50 5.00 7.50

PDACell Culture,

PDA

AM

Q

NH

3

HyP

ro

His

Asn

Tau

Ser

Gln

Arg

Gly

Asp

Glu

Thr

Ala

GA

BA

Pro

HyL

ys1

HyL

ys2

AA

BA

Orn

Der

iv P

eak

Cys

Lys

Tyr

Met

Val

NV

a

Ile

Leu

Phe

Trp

EU

0.00

800.00

1600.00

Minutes0.00 2.50 5.00 7.50

Cell Culture, TUV

Cell Culture, FLRCell Culture, FLR

AM

Q

NH

3H

yPro

His

Asn T

au Ser G

lnArg G

ly Asp

Glu

Thr

Ala GABA Pro

HyL

ys1

HyL

ys2

AABA

Orn

Der

iv P

eak

Cys

Lys

Tyr

Met Val

NVa

Ile

Leu

Phe T

rp

AU

0.00

0.05

0.10

Minutes0.00 2.50 5.00 7.50


H-ClassUPLC

System CD– User’s Guide

– Empower projects

AccQ●Tag Ultra Chemistry Package– AccQ●Tag Ultra Eluent A Concentrate

– AccQ●Tag Ultra Eluent B

– AccQ●Tag Ultra Column

– AccQ●Tag Ultra Derivatization Kit

– Amino Acid Hydrolysate Standard

Low flow inlet tubing

Column in-line filter kit

Comparison of Application Kit

System CD– User’s Guide

– Empower projects

AccQ●Tag Ultra Chemistry Package– AccQ●Tag Ultra Eluent A Concentrate

– AccQ●Tag Ultra Eluent B

– AccQ●Tag Ultra Column

– AccQ●Tag Ultra Derivatization Kit

– Amino Acid Hydrolysate Standard

Low flow inlet tubing

Column in-line filter kit

150mm column stabilizer

2μL sample loop


ACQUITY UPLC H-Class and H-Class BioAmino Acid Analysis System

Turn-key UPLC® Amino Acid Analysis application – Optimized for the ACQUITY UPLC H-

Class Systems– Dedicated QC-tested column and

reagents – Application-specific Performance

Installation Test and Training– Same result day-to-day,

instrument-to-instrument, lab-to-lab, around the world

Target Applications– Protein and peptide identification

and quantitation– Monitoring cell culture media– Analysis of Foods and Feeds


H-Class Amino Acid Analysis SystemCell Culture Amino Acids

AM

Q

NH

3H

yPro

His

Asn T

au Ser G

lnArg G

ly Asp

Glu

Thr

Ala

GABA Pro

HyL

ys1

HyL

ys2

AABA

Orn

Der

iv P

eak

Cys

Lys

Tyr

Met Val

NVa

Ile

Leu

Phe T

rp

AU

0.00

0.05

0.10

Minutes0.00 2.50 5.00 7.50


Retention Time Reproducibility


Area Reproducibility


Quantitative LinearityDerivatized Amino Acids

0.05 0.075 1.0 2.5 25 50AMQ 6033932 6020972 6101437 6056268 5069175 3648294NH3 4064 4810 9094 14396 86830 155693His 167 304 4064 9618 104287 204119Ser 334 330 4052 9459 102749 199653Arg 230 320 3994 9516 102770 201123Gly 591 682 4260 9608 100540 196412Asp 211 272 4053 9704 101645 190396Glu 238 303 3988 9330 100969 190784Thr 238 329 4115 9617 104228 201799

CM Cys 197 310 4110 9726 105289 211792Ala 269 336 4029 9447 102180 195455

PE Cys 240 339 4281 10014 109782 224055Pro 219 308 3807 8916 96538 187201

D-Peak 94086 84767 94312 89565 62635 46575Cys 172 262 3566 8315 91527 179095Lys 334 499 6810 15852 173792 329877Tyr 217 320 4177 9874 106770 209182Met 176 268 4049 9354 104998 204492Val 274 374 4127 9765 105725 204246NVa 219 331 3893 8911 99315 198500Ile 373 435 4323 9899 106378 205866

Leu 225 300 4084 9521 103758 201006Phe 237 348 4312 9970 106180 208031

Peak Name

Conc (pmol/uL), Ave Peak Areas

y = 4098.3x + 54.943R² = 0.9999

0

50000

100000

150000

200000

250000

0 5 10 15 20 25 30 35 40 45 50

His, Area vs. Conc


Analysis of Protein Hydrolysates

Purified Proteins or Simple Pharmaceutical Formulations

Confirmation of Protein Identity

Determination of Protein Amount

Requirements– Unequivocal peak identification

– Two order-of-magnitude linear dynamic range

Limitations– Destruction of Tryptophan, Asparagine, and Glutamine

– Variable, but poor recovery of sulfur-containing amino acids

– Other variables in hydrolysis procedure

– Matrix interference

– Compositional accuracy on the order of 5%


Analysis of Protein HydrolysateBovine Serum Albumin

NH

3

His

Ser

Arg

Gly

Asp

Glu

Thr

Ala

Pro

Der

iv P

eak

Cys

Lys

Tyr

Met

Val

Ile

Leu

Phe

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

Minutes1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20


Quantitative LinearityBSA Hydrolysate, 20-1000μg/mL

NH

3

His S

er

Arg

Gly

Asp

Glu

Thr A

la

Pro

Der

iv P

eak

Cys

Lys

Tyr

Met

Val

Ile

Leu

Phe

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

Minutes1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00


Quantitative LinearityBSA Hydrolysate, 20-1000μg/mL

NH

3

His S

er

Arg

Gly

Asp

Glu

Thr A

la

Pro

Der

iv P

eak

Cys

Lys

Tyr

Met

Val

Ile

Leu

Phe

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

Minutes1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Asp Glu

Thr

Ala


Analysis of Alkylated Cysteines

AM

Q

NH

3

His

Ser A

rgG

ly Asp

Glu

Th

r

CM

Cys

Ala

PE C

ys

Pro

Der

iv P

eak

Cys

Lys

Tyr

Met

Val

NV

a

Ile

Leu

Phe

AU

0.00

0.05

0.10

Minutes0.00 2.50 5.00 7.50


Analysis of Cell Culture Media

Growing cell culture media

Measure free amino acids rather than hydrolysates– Nutritional status

– Waste products

Larger array of amino acids

Measure composition of media batches

Measure changes over time


H-Class Amino Acid Analysis SystemCell Culture Amino Acids

AM

Q

NH

3H

yPro

His

Asn T

au Ser G

lnArg G

ly Asp

Glu

Thr

Ala

GABA Pro

HyL

ys1

HyL

ys2

AABA

Orn

Der

iv P

eak

Cys

Lys

Tyr

Met Val

NVa

Ile

Leu

Phe T

rp

AU

0.00

0.05

0.10

Minutes0.00 2.50 5.00 7.50

Additional Amino Acids


Ser

Gln

Arg

Gly A

sp

Glu

Thr

Ala

Minutes2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00

Cell Culture Sample

1 day

3 day

6 day


Amino Acid AnalysisFoods and Feeds

Nutritional Content– Total amount of amino acids

– Optimize blended feeds

Identification/Adulteration– Unusual amino acids

– Marker amino acids

– Proprietary constituents

Process monitoring– Changes during processing

– Changes during storage


Amino Acid AnalysisClasses of Analysis

Components of proteins - “Bound” amino acids

Metabolic intermediates - “Free” amino acids– Building blocks for protein

– Source of nitrogen for the synthesis of compounds such as nucleotide bases or vitamins

– Biomarkers in metabolic and genetic studies

– Surplus amino acids are used as metabolic fuel


Amino Acids as Biomarkers

Free Amino Acids– Nutrients

– Metabolic intermediates

– Waste products

Qualitative and Quantitative Profile– Genotype of food

– Origin

Practical Application– Process Monitoring

– Identity and adulteration


H-Class Amino Acid Analysis SystemFree/Cell Culture Amino Acids

AM

Q

NH

3H

yPro

His

Asn T

au Ser G

lnArg G

ly Asp

Glu

Thr

Ala

GABA Pro

HyL

ys1

HyL

ys2

AABA

Orn

Der

iv P

eak

Cys

Lys

Tyr

Met Val

NVa

Ile

Leu

Phe T

rp

AU

0.00

0.05

0.10

Minutes0.00 2.50 5.00 7.50


AM

Q

NH

3H

yPro

His Asn Ta

u

Ser

Gln A

rgG

ly

Asp G

lu Thr

Ala

GA

BA

Pro H

yLys

1H

yLys

2

AA

BA

Orn

Der

iv P

eak

Cys

Lys

Tyr

Met

Val NV

a

Ile Leu P

heTr

p

AM

Q

NH

3

Asn

Ser

Gln

Arg

Gly

Asp

Glu

Thr

Ala

GA

BA

Pro

AA

BA

Orn

Der

iv P

eak

Cys

Lys

Tyr

Met V

alN

Va

Ile Leu Phe

Trp

Minutes1.00 1.50 2.00 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

0.1

0 A

U0.1

0 A

U

Free Amino Acid Standard

Orange Juice

Free Amino AcidsOrange Juice


AM

Q

NH

3

His

Asn

Tau Ser

Gln

Arg

Gly Asp G

lu Thr

Ala

GA

BA

Pro

AA

BA

Orn

Der

iv P

eak

Lys

Tyr

Met V

alN

Va

Ile Leu

Phe

Trp

AM

Q

NH

3

Asn

Tau

Ser

Arg

Asp

Glu

Thr

Ala

GA

BA

Der

iv P

eak

Lys

Tyr

Val

NV

a

Ile Leu

Phe Trp

AM

Q

NH

3

His

Asn

Ser

Gln

Arg

Gly

Asp

Glu

Thr

Ala G

AB

A

Pro

AA

BA

Orn

Der

iv P

eak

Cys Ly

sTy

rM

et Val

NV

a

Ile Leu Phe

Trp

Minutes1.00 1.50 2.00 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

0.1

0 A

U0.1

0 A

U0.1

0 A

U

Grape Juice

Apple Juice

Orange Juice

Free Amino AcidsDifferent Juices


AM

Q

NH

3

His

Asn

Tau S

erG

lnA

rgG

ly Asp G

lu Thr

Ala

GA

BA

Pro

Orn

Der

iv P

eak

Lys

Tyr

Met V

alN

Va

Ile Leu

Phe

Trp

AM

Q

NH

3

His Asn

Tau S

erG

lnA

rgG

ly Asp G

lu Thr

Ala

GA

BA

Pro

Orn

Der

iv P

eak

Lys

Tyr

Met V

alN

Va

Ile Leu

Phe

Trp

Minutes1.00 1.50 2.00 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

0.1

0 A

U0.1

0 A

U

Grape Juice #1

Grape Juice #2

Free Amino AcidsDifferent Brands of Grape Juice


AM

Q

NH

3H

yPro

His Asn Ta

u

Ser

Gln A

rgG

ly

Asp G

lu Thr

Ala

GA

BA

Pro H

yLys

1H

yLys

2

AA

BA

Orn

Der

iv P

eak

Cys

Lys

Tyr

Met

Val NV

a

Ile Leu P

heTr

p

AM

Q

NH

3

Tau

GA

BA

Der

iv P

eak

Val

NV

a

Minutes1.00 1.50 2.00 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

0.1

0 A

U0.1

0 A

U


Energy Drink

Free Amino AcidsPopular Energy Drink


AM

Q

NH

3H

yPro

His Asn Ta

u

Ser

Gln A

rg Gly

Asp G

lu Thr

Ala

GA

BA

Pro H

yLys

1H

yLys

2

AA

BA

Orn

Der

iv P

eak

Cys

Lys

Tyr

Met

Val NV

a

Ile Leu P

he Trp

AM

Q

NH

3

Asn

Tau S

erG

lnA

rg Gly

Asp

Glu

Thr Ala

Pro

Orn

Der

iv P

eak

Lys

Val

NV

a

Ile Leu Trp

AM

Q

NH

3H

yPro

His

Asn

Tau

Ser

Gln Arg Gly

Asp

Glu

Ala

Pro

Orn

Der

iv P

eak

Lys

Val

NV

a

Leu

Trp

Minutes1.00 1.50 2.00 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

0.1

0 A

U0.1

0 A

U0.1

0 A

U


Fat free milk

Reconstituted Infant formula

Free Amino AcidsAdditives in Infant Formula


Amino Acid AnalysisFoods and Feeds

Nutritional Content– Total amount of amino acids

– Proportions of essential amino acids

– Optimize blended feeds


Food and Feed Hydrolysate Method Different Separation Conditions

AM

Q

NH

3

Cya

His

Tau

Ser Arg

Gly A

sp Met

SO

2

Glu T

hr

Ala

Pro

AABA

Der

iv P

eak

Cys

Lys

Tyr

Met Val

NVa

IIe

Leu

Phe

AU

0.00

0.05

0.10

Minutes0.00 2.50 5.00 7.50

Different Gradient TableHigher Temperature


AM

Q

NH

3C

ya His Ta

u

Ser Arg

Gly

Asp Met

SO

2

Glu Th

r

Ala Pro

AA

BA

Der

iv P

eak

Cys Ly

sTy

rM

etV

al NV

a

Ile Leu

Phe

AM

Q

NH

3

His Ser

Arg Gly

Asp G

lu

Thr

Ala Pro

Der

iv P

eak

Cys Ly

sTy

rM

et Val

NV

a

Ile Leu

Phe

AM

Q

NH

3

His Ser

Arg Gly

Asp G

lu

Thr

Ala Pro

Der

iv P

eak

Cys Ly

sTy

rM

et Val

NV

a

Ile Leu

Phe

AM

Q

NH

3

His Ser

Arg Gly Asp G

lu

Thr

Ala

Pro

Der

iv P

eak

Cys Ly

sTy

rM

et Val

NV

a

Ile Leu

Phe

Minutes1.00 1.50 2.00 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

0.1

0 A

U0.1

0 A

U0.1

0 A

U0.1

0 A

U

Food and Feed Amino Acid Standard

Swine feed

Poultry feed

Soy Meal Hydrolysate

Amino Acid AnalysisHydrolyzed Feeds


Hydrolysis of Feeds

Acid Hydrolysis– 6N/Constant Boiling HCl, 105° for 24 hours– Sample suspended in 10-100-fold weight excess of acid– Sealed and heated– Best method for complex samples– Destroys some amino acids

Oxidation before hydrolysis for cysteine and methionine

Alkaline hydrolysis for tryptophan

Cumbersome– Long hydrolysis time– Bulky apparatus requirements, especially for multiple samples


Microwave Hydrolysis

Faster cycle time

Controlled hydrolysis conditions

Automated sample handling


WorkflowHydrolysis and Analysis


Microwave Acid HydrolysisPerformic Acid Oxidation

NH

3

Cya H

is

Tau Ser Arg

Gly

Asp M

etSO

2

Glu Thr

Ala Pro

AABA

Der

iv P

eak

Cys

Lys

Tyr

Met Val

NVa

IIe

Leu

Phe

TrpAU

0.00

0.05

0.10

0.15

AU

0.00

0.05

0.10

0.15

0.20

AU

0.00

0.05

0.10

0.15

AU

0.00

0.02

0.04

0.06

Minutes1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20


Oxidized Soy Flour

Oxidized Turkey Starter

Oxidized Nutritional Supplement


Complete Amino Acid AnalysisTurkey Starter

NH

3

Cya H

is

Tau Ser Arg

Gly

Asp M

etSO

2

Glu Thr

Ala Pro

AABA

Der

iv P

eak

Cys

Lys

Tyr

Met Val

NVa

IIe

Leu

Phe

Trp

AU

0.00

0.05

0.10

0.15

AU

0.00

0.05

0.10

0.15

0.20

AU

0.00

0.02

0.04

0.06

0.08

AU

0.00

0.05

0.10

0.15

Minutes1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20


Alkaline Hydrolysis

Acid Hydrolysis

Oxididation withAcid Hydrolysis


Application ClassesSame Separation Conditions

AM

Q

NH

3H

yPro

His

Asn T

au Ser G

lnA

rg Gly Asp

Glu

Th

r

Ala

GA

BA Pro

HyL

ys1

HyL

ys2

AA

BA

Orn

Der

iv P

eak

Cys

Lys

Tyr

Met Val

NV

a

Ile

Leu

Phe T

rp

AU

0.00

0.05

0.10

Minutes0.00 2.50 5.00 7.50

Cell Culture, TUV

AM

Q

NH

3

His S

er Arg

Gly A

sp

Glu

Th

r Ala

Pro

Der

iv P

eak

Cys

Lys

Tyr

Met

Val

NV

a Ile

Leu

Phe

AU

0.00

0.05

0.10

Minutes0.00 2.50 5.00 7.50

Hydrolysate, TUV

AM

Q

NH

3

His

Ser A

rgG

ly Asp

Glu

Th

r

CM

Cys

Ala

PE C

ys

Pro

Der

iv P

eak

Cys

Lys

Tyr

Met

Val

NV

a

Ile

Leu

Phe

AU

0.00

0.05

0.10

Minutes0.00 2.50 5.00 7.50

Alkylated Cysteines, TUV

AM

Q

NH

3

Cya

His

Tau

Ser Arg

Gly A

sp Met

SO

2

Glu T

hr

Ala

Pro

AABA

Der

iv P

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Feed Hydrolysate, TUVDifferent Method


ACQUITY UPLC H-Class and H-Class BioAmino Acid Analysis System

Turn-key UPLC® Amino Acid Analysis application – Optimized for the ACQUITY UPLC H-

Class Systems– Dedicated QC-tested column and

reagents – Application-specific Performance

Installation Test– Same result day-to-day,

instrument-to-instrument, lab-to-lab, around the world

Target Applications– Protein and peptide ID and

quantitation– Monitoring cell culture media– Analysis of Foods and Feeds

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Introduction to the ACQUITY UPLC H-Class Amino Acid ... · r Met Val NVa Ile Leu Phe Trp Minutes...

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