of high‐speed uhplc high resolution orbitrap ms in the...

Application of High Speed UHPLCApplication of High‐Speed UHPLC and High‐Resolution Orbitrap‐MS in the Analysis of Pharmaceutical Compounds

Christian Huber

ThermoScientific Lunch Symposium @ HPLC2011

Citius,altius,

f tifortius

Department of Molecular BiologyDivision of Chemistry and Bioanalytics 2

Cs. Horváth, LC GC Magazine, 9, 1996, 622‐623

Ultrafast Analysis of ThyroidUltrafast Analysis of Thyroid Hormone Degradation Products

Thyroxin manufacturer: peptido1 2 3 4 5 6 7peptido

1 2 3 4 5 6 7


active substancesactive substances

Thyroxin as active pharmaceutical ingredient

I O

I

OH

NH2

Thyroxin, T4

OH

I

IOH

O

I

Liothyronine T3

I

OH

O

IOH

O

NH2

Liothyronine, T3 O

• Synthetic Thyroxin needs to be carefully

checked for s nthesis b prod cts or stress

• Worldwide up to 1 Billion people

h l k f Th i (WHO 2003) checked for synthesis by‐products or stress‐

induced degradation products, respectively.

• Development of a high troughput MS‐

show a lack of Thyroxine (WHO, 2003).

• Hypothyroidism requires a lifelong treatment with Thyroxin

compatible analysis method.

• Structure elucidation of impurities byhigh‐resolution mass spectrometry.

treatment with Thyroxin.

• Thyroxin acts as a prohormone ofits active form Liothyronine.


high resolution mass spectrometry.y

Thermal stressing as a model for storage

Model analytes for thermal stressing:

O

I

T2/T3/T4O

I

HO

I

I

NH2

COOH

I

r-T2/r-T3

I

O

HO

I NH2

COOH

I

TriPropO

HO

I

I COOH

I

Thyroxin kept at 40°C for 6 monthsand finally stressed for 16 h at 60 °C.

DiAc/TriAc/TetraAc

O

I

HO

I

ICOOH

TetraFAO

I

I

HO

I

I COOH


I

Instrumental requirements

Accela 1000 bar UHPLC system:

• 1000 bar pressure limit pump

• 1000 µL/min maximal flow rate

Accela 1250 bar UHPLC system:

• 1250 bar pressure limit pump

• 2000 µL/min maximal flow rate• 1000 µL/min maximal flow rate

• Hypersil GOLD column, 100 x 2.1 mm,

1.9 µm particles• 20 Hz UV Detector

• 2000 µL/min maximal flow rate

• Hypersil GOLD column, 100 x 2.1 mm,

1.9 µm particles• 80 Hz UV Detector


• 20 Hz UV Detector • 80 Hz UV Detector

The route to high‐throughput analysis

• YMC C18 Pro, 150 x 4.6 mm, 5µm

• 38 min acetonitrile gradientT4

DiAc

TriAc TriProp

TetraAcT2• 4.5 mM Phosphate buffer

• 900 µL/min, UV @225 nm.

rT3

rT2 T3TetraAc

TetraFA

Application of UHPLC

T2

Application of UHPLC using:

1) Gradient volumeprinciple

0.0 10.0 30.0 40.0 50.0 min

principle

2) Gradient fine tuning including optimization

DiAcTriProp

of separation temperture

T2

• Hypersil GOLD, 100 x 2.1 mm, 1.9 µm

rT2

rT3

T3T4 TriAc

p

TetraAc

TetraFA

0 0 0 5 2 5 min1 5 2 01 0

• 2.1 min acetonitrile gradient,

• 0.1 % Formic acid

• 1000 µL/min, UV @ 280 nm


0.0 0.5 2.5 min1.5 2.01.0

UHPLC and the gradient‐volume principle

18.0

nm

1

2

6

VG = F tG= konst.VG, gradient volumeF volumetric flow rate

mA

U @

280

2

34

57

89 10

9.0

F, volumetric flow ratetG, gradient time

5 min at 30% ACN, 30‐70% ACN in 37 min, 225 µl/min 270 bar

0.0 20.0 40.0 min

0.0

In practice: halving of gradient time

requires doubling of flow rate !225 µl/min, 270 bar.

12.0

0 n

m 1

2

6

7• Hypersil GOLD column,

mA

U @

280 2

34

5

78

9 106.0 100 x 2.1 mm, 1.9 µm

• Accela UHPLC system

• 1000 bar pressure limit

1.25 min at 30% ACN, 30‐70% ACN in 9.25 min, 900 l/ i 920 b

0.0 5.0 10.0 min

0.0• 1000 bar pressure limit

•Maximal flow rate

1000 µL/min


900 µl/min, 920 bar.

Optimized UHPLC‐MS method0

nm

25.06

7 8

UV-detection • LTQ‐Orbitrap XL mass spectrometer

• Maximum resolution of 100,000

4 S / t R 15 000

mA

U @

280 1 2

3

4

7 89105

• 4 Scans/s at R = 15,000

• Mass accuracy < 1 ppm

106

0.0

10 0

0 0.5 2.5 min1.5 2.01.0

y [c

ou

nts

]x1 10.0 negESI-MS detection

20‐fold reduction of nal

in

ten

sity

total analysis time from 60 to 3 min !

Sig

n

0.00.0 0.5 2.5 min1.5 2.01.0

30‐80% acetonitrile in 0.1 % formic acid i 2 1 i 1000 l/ i 620 b 60°C


in 2.1 min, 1000 µl/min, 620 bar, 60°C.

Reproducibility of the ultrafast method

3.50% Retention timePeak area (UV)

2.50%

3.00%Peak area (UV)Peak area (MS)Mass deviation ppm

1.50%

2.00%

0 50%

1.00%

0.00%

0.50%

Day1 Day2 Day3 Day1-3Day1 Day2 Day3 Day1 3

∑ 60 measurements

0.5%=0.01 min (0.6 s) in 2 min, 0.5 ppm =0.00035 Da in 700 Da!


0.5% 0.01 min (0.6 s) in 2 min, 0.5 ppm 0.00035 Da in 700 Da!

High‐end separations reaching instrumental limits

25.0n

m• System Accela I• 30‐80% ACN + 0.1 % FA in 2.1 min• Maximum flow rate of 1000 µL/min• Column backpressure 620 bar (1000

6

78

mA

U @

280

n • Column backpressure 620 bar (1000 bar pressure maximum)

• Column temperature 60 °C

1 2

3

4

8

9105

0.00.0 0.5 2.5 min1.5 2.01.0

m

15.0

nm

Gradient volume principle!

mA

U @

280

n

• System Accela II• 30‐80% ACN + 0.1 % FA in 1.05 min

0.00.0 0.25 1.25 min0.75 1.00.5

m • Maximum flow rate of 2000 µL/min• Column backpressure 1210 bar

(1250 bar pressure maximum)• Column temperature 60 °C


Column lifetime under UHPLC conditions

Hypersil GOLD

column number

Total injection number without sigificant loss in column efficiency

Blankinjections

StandardInjections

(20 – 200 ppm)

Thyroid hormoneInjections(2000 ppm)

column efficiency( pp ) ( pp )

1 2050 200 450 1400

2 1200 500 400 300

3 3300 2500 400 400

Average 2180 1060 420 700

26 6Column 1 UHPLC di i f ll

0

132 3

4

57

9 1081

Column 1 after 2050 injections

UHPLC conditions of all measurements:

•Hypersil GOLD, 100 x 2.1 mm; 1.9 µm•Maximum flow rate of 1000 µL/min•Column backpressure 620 barm

AU

]

0

0

1.5 3.0 min

•Column backpressure 620 bar•Column temperature 60 °C

A well packed UHPLC column300

al i

nte

nsi

ty [

1 2 34 5 6

After 3 injectionsAfter 3100 injections

Column 2

tolerates more than 2000 injections

under UHPLC conditions!

0

0

150

1 0 2 0 3 0

Sig

na After 3100 injections


0 1.0 2.0 3.0

Higher information content: interfacing to high‐resolutionmass spectrometrymass spectrometry


Instrumental platforms

Accela‐Exactive:

• max. resolution of 100.000

• 1 scan per s @ R=100.000

Accela‐LTQ Orbitrap XL:

• max. resolution of 100,000

• 4 scans/s @ R=15,000

• 10 scans per s @ R=10.000

• < 1 ppm mass accuracy

• No MSn possibility

• 0.5 scans/s @ R=100.000

• < 1 ppm mass accuracy

• MSn possibility


p y p y

Analysis of thermally stressed Thyroxin

1.55 min

Base peak chromatogram

Thyroxin

1 2 3 min0

3

4

5 78

9

10817 69762.65

Scan Nr. 2

6

731 65

Scan Nr. 12

1.53 1.55 1.58 min

1

10

112817.69

745.6712 762.65

774.65745.67

731.65

775 850 m/z700

745.67Scan Nr. 6

750 800 m/z700

750 800 m/z700

I it tt th 300 d t t d !


Impurity pattern: more than 300 masses detected !

From accurate mass to structure

www.ich‐bin‐dann‐mal‐schlank.de/magazin_v2/wp‐content/uploads/2011/06/IBDMS‐Waage‐help‐450x298.jpgh b d / h bl /


thumbs.dreamstime.com/thumblarge_401/124292109408v6qR.jpg

Simplification of impurity patternStep 1: Generation of molecular formulas from accurate masses.

f id d ki l i312 candidates

Software‐aided ranking applying:

• limited elements (C,H,O,N,I,Na)•< 2ppm mass accuracy Nit l i it

even‐ 0.3209.5C14H8O3NI4

e‐Δ ppmRDBMolecular formula

• Nitrogen rule , ion parity

m/z =

Accurate mass:

odd‐ 1.9964.0CHO15N8I3

odd1.48110.0C12H6O2N4I4

i f i l d d i l i

745.66860

[M-H]-

745.66774

Comparison of simulated and experimental isotope pattern:

[M-H]-

745.66860

267 candidates left749 m/z745 747 749 m/z745 747


Simplification of impurity patternStep 2: Assembly of molecular formulas deriving from different species of the same compound.

267 candidates

p

Ordering of molecular formulasby retention time

RT: 1.78 min

[M‐H]‐ Found impurityC14 H7 O4 I4

[M‐H‐CO2]‐

[2M‐2H+Na]‐

[2M‐H]‐

[ ]

Sodium adductC28 H14 O8 I8 Na

Gas phase dimerC28 H15 O8 I8

ESI‐fragmentC13H7O

2I4

p y14 7 4 4

Identification of related molecularformulas

[ 2] g13 7 2 4

180 candidates left[M‐H]‐ Σ peak areaC14 H7 O4 I4

Assembly andsummation of peak area


Simplification of impurity patternStep 3: Check of chemical plausibility of molecular formulas NH2

COOR

Thyroxin

180 candidatesplausibleThermal

stressing

COO

HO

C15H10O5NI4O

I

INH2

Thyroxin

NH2

COOR

HO OH

HOnot plausible

Hydrate!

O2

hardly oxidizable

I

HO I COO

oxidizable

For a specific number of carbon atoms in a molecularformula only a limited number of oxygen is plausible!

C15H10O7NI4

ycore structure side chain

formula, only a limited number of oxygen is plausible!(Erlenmeyer rule)

Result of step 1‐3:

145 candidates left

•Massive reduction of impurity number: 312 145

•Minimal loss of overall peak area : 100 % 96 %


Impurity distribution according to reaction pathways

Degradation pathways:

Deiodination Oxidation / side chain degradation + deiodinationOxidation / side chain degradation

Quantitative distributionaccording to relative peak area

Qualitative distributionaccording to number of compounds

MonomersDimers

MonomersDimers

O id ti d id h i d d tiR ti b t d di

Monomers

Dimers

Oxidation and side chain degradationrepresented by 84 % of peak area.

Ratio between monomers and dimers: 46 to 54 %.

Comparison of absolute peak areas:Comparison of absolute peak areas:

• 20 main impurities make up 91 % of the total impurity peak area

• 16 of the main impurites derive from oxidation / side chain degradation


16 of the main impurites derive from oxidation / side chain degradation

Conclusions

Small particles enable ultra‐fast separationsp pat high flow rates (UHPLC).

A t d t ti i i di bl f Accurate mass detection is indispensable for the characterization of unknown impurities.

High‐resolution fragmentation is a powerful toolfor structure elucidation.

The coupling of UHPLC and HRMS opens up new perspectives in pharmaceutical quality controlperspectives in pharmaceutical quality control(productivity, cost & resource control).


Acknowledgments

Instrumental support:


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