liquid chromatography (lc) - uz leuven · 2009-02-16 · liquid chromatography (lc) • technique...

Liquid Chromatography (LC)• technique that is useful for separating ions or

molecules that are dissolved in a solvent • the mixture to be separated is loaded onto the

top of the column followed by more solvent • different components in the sample mixture pass

through the column at different rates due todifferences in their partioning behavior betweenthe mobile liquid phase and the stationary phase

High-Performance LiquidChromatography (HPLC)

• is a form of liquid chromatography• HPLC instruments consist of

– reservoir of mobile phase– pump– an injector– separation column– detector

• compounds are separated by injecting a plug of the sample mixture onto the column

What is Mass Spectrometry (MS)?

• measuring the molecular weight (MW)• for biomolecules:

– accuracy of 0.01% of the total molecular weight

– sufficient to allow minor mass changes to be detected:

• e.g. the substitution of one amino acid for another

Where are Mass Spectrometers Used?

• Biotechnology: proteins, peptides, oligonucleotides

• Pharmaceutical: drug discovery, pharmacokinetics, drug metabolism

• Clinical: neonatal screening, haemoglobin analysis, drug testing

• Environmental: PCBs, water quality, food contamination

How Does a Mass Spectrometer Work?

• Mass spectrometers can be divided into three fundamental parts:– the ionisation source– the analyser– the detector

Sample Introduction

• The sample can be inserted:– directly into the ionisation source– undergo some type of chromatography en

route to the ionisation source • high pressure liquid chromatography (HPLC)• gas chromatography (GC) • capillary electrophoresis (CE)

Methods of Sample Ionisation

• Ionisation methods include the following:– Atmospheric Pressure Chemical Ionisation

(APCI)– Electrospray Ionisation (ESI)– Atmospheric Pressure Photo Ionisation (APPI)– Matrix Assisted Laser Desorption Ionisation

(MALDI)– Etc. etc.

Electrospray Ionisation• sample is dissolved in a polar, volatile solvent

and pumped through a narrow, stainless steel capillary

• high voltage of 3 or 4 kV is applied to the tip of the capillary

• sample emerging from the tip is dispersed into an aerosol of highly charged droplets

• aided by a co-axially introduced nebulising gasflowing around the outside of the capillary

Analysis and Separation of Sample Ions

• main function of the mass analyser is:– to separate the ions formed in the ionisation

source – according to their mass-to-charge (m/z)

ratios • mass analysers have different features:

– m/z range that can be covered– the mass accuracy– achievable resolution

Detection and Recording of Sample Ions

• The detector:– monitors the ion current– amplifies the ion current– generates mass spectra– The m/z values of the ions are plotted against their

intensities• The m/z values of the ions are plotted against

their intensities:– shows the number of components in the sample– the molecular weight of each component– the relative abundance of the various components

Tandem Mass Spectrometry

• structural information about a compound – fragment specific sample ions inside the mass

spectrometer– identify the resulting fragment ions

• the two analysers are separated by a collision cell into which an inert gas (e.g. argon, xenon) is admitted to collide with the selected sample ions and bring about their fragmentation

Selected/multiple reaction monitoring

• MRM:– both of the analysers are static – user-selected specific ions are transmitted through

the first analyser– user-selected specific fragments arising from these

ions are measured by the second analyser• used to confirm unambiguously the presence of

a compound in a matrix • not only a highly specific method but also has

very high sensitivity

http://marketing.appliedbiosystems.com/mk/submit?_m1_charset=UTF-8&_A=41089&_D=29047&_V=8#

Urinary cortisol

Poor specificity and recovery of urinary free cortisol as determined by the Bayer ADVIA Centaur extraction method.

Commercial radioimmunoassays do not measure urinary freecortisol accurately and should not be used for physiologicalstudies.

How Much "UFC" Is Really Cortisol?

Lack of specificity of urinary free cortisol determinations: why does it continue?

Urinary cortisol by LC-MS/MS

• Centrifuge urine to remove all sediment• Transfer in borosilcate tube• Add deuterated internal standard• Add methylene chloride and vortex• Centrifuge and remove aqueous layer• Wash methylene chloride fraction:

– With sodium hydroxyde– With hydrochloric acid– With water

Urinary cortisol by LC-MS/MS

• Evaporate under nitrogen• Reconstitute with methanol-water/estriol• Centrufuge• Transfer to autosampler vials

Urinary cortisol by LC-MS/MS

• Tripe-quadrupole mass spectrometer (API 2000)

• ESI source• Reverse phase colum with precolmn filter• Methanol/water mobile phase• Positive ion mode• 3 minutes per sample

ESI mass spectrum (Q1 scan) of cortisol in positive mode, showing the [M +1] molecular ion

product ion scan showing the major transition fragment at m/z 121

Conclusions UFC• best screening test for hypercortisolism• challenge for the laboratory:

– many compounds– cortisol metabolites– polarities and chemical structures similar to those of

cortisol• HPLC-UV methods ??

– interfering compounds still require separation– prolongs chromatographic run time– decreases throughput

Conclusions UFC (2)

• LC-MS/MS ??– More specific

• Nebulizer vaporizes and ionezes the compounds• Eliminates the need voor derivatisation (GC-MS)• Collision induced dissociation• Q2 selects (specific) fragments

– More sensitive• Cleaner chromatograms• Eliminating interferences• Shorter run times (troughput increases)

Vitamin D

TABLE 1. 25OHD assay methodology and normal range

Laboratory MethodologyNormalrange

A Acetonitrile extraction followed by in-house RIA 10–55 ng/mlB DiaSorin RIA 10–40 ng/mlC Acetonitrile extraction followed by DiaSorin RIA 8–38 ng/mlD Chemiluminescent assay 20–57 ng/mlE Acetonitrile extraction followed by DiaSorin RIA NAF Chemiluminescent assay 6–54 ng/mlG Chemiluminescent assay 10–68 ng/ml

H Ethyl acetate extraction followed by normal phaseHPLC

NA

Materials

• HPLC: Agilent 1100– Quaternary pump– Vacuum degasser– Temperature controlled autosampler– Temperature controlled column oven

• Tandem mass spectrometer: API 3000– ESI– “Analyst” software– MRM mode with m/z 402>383.5 for 25-OH-D3

Sample preparation

• 100 µL sample• 75 µL IS in methanol:propanol 80:20 (protein

precipitation)• 10 sec vortex• 500 µL hexane (extraction)• 10 sec vortex• centrifuge• evaporate in autosampler vials• Reconstitute in methanol/water 70:30

LC-MS/MS

• BDS C8 reversed-phase column• inject 50 µL• methanol gradient• 8 min total run time• positive ion mode• hexadeuterated 25-OH D3 as an IS

383.5 corresponding to the molecular ion with a loss of water

Analytical performance

• LOQ (signal-to-noise ratio >10): 1.6 ng/ml• Imprecision:

– 6.2 % CV at 6.4 ng/ml– 3.5 % CV at 14 ng/ml– 5.2 % CV at 30.4 ng/ml

• Spiking revcovery 94% - 108%• Linearity by dilution: 89% - 113%• Possible interfering compounds with same MW

were considered unimportant or tested

Testosterone

Immunoassays for testosterone in women: better than a guess?

David A. Herold*

Robert L. Fitzgerald

VA San Diego Healthcare System and

University of California, San Diego

“In the case of testosterone, the immunoassays do not work in healthyfemales and fail miserably when used inpotentially diseased females”

Clinical Chemistry 49, No. 8, 2003

Editorial: Serum TestosteroneAssays—Accuracy Matters

“Rigorous attention to the accuracy of many hormone assays, includingTassays, has lagged behind and in someinstances, been overlooked”

J. Clin. Endocrinol Metab, February 1, 2004; 89(2): 520 –524

M. Matsumoto and W. J. Bremner

Serum testosterone assays:role

In males:• confirm the diagnosis of hypogonadism• evaluate boys with delayed or

precocious puberty• monitor the adequacy of T therapy

Serum testosterone assays:role

In females:• evaluation of hyperandrogenism

– idiopathic hirsutism– congenital adrenal hyperplasia– polycystic ovarian syndrome– androgen-secreting ovarian or adrenal

tumors• diagnose androgen deficiency

T assays: history

• 1970: development of RIAs for T– organic extraction– chromatographic separation

• T assays of today:– more sensitive and specific– require smaller quantities of serum– do not involve extraction or chromatography– performed more rapidly– less cost– automated platforms using nonradioactive

methods

T assays of today

• performed with proprietary reagents• reference ranges provided by manufacturer

(not corresponding with standard referencetexts)

• limited published validation data• approval of these methods by regulatory

agencies for clinical use is primarily based onnoninferiority comparison against previouslyapproved assays

Reasons for differences

• matrix effect: certain compounds present inserum interfere with the immunoassay

• cross-reactivity with structurally relatedsteroids

limit of detection and functional sensitivityparticularly important for the determination of low (1.7 nmol/L) and very low (0.17 nmol/L) testosterone concentrations

Conclusions Wang

• In this study, the DPC-RIA and RocheElecsys methods for determining serum T levels show the closest correlation withvalues determined by LC-MSMS.

• Without modification, none of the automatedmethods are currently acceptable for the measurement of T in the serum of normalfemales or children.

Conclusions Taieb et al.

• “None of the immunoassays tested wasreliable enough for the investigation of the very low and low testosterone concentrations(0.17–1.7 nmol/L) expected in sera fromchildren and women. These assays aretherefore unlikely to be useful for diagnosis, follow-up of sexual differentiation, or generaluse in pediatric surveys.”

• “In addition, we consider two of these assays(ACS-180 and AutoDelfia) questionable at the low limit of testosterone concentrations in men.”

RIA Biosource versus GC-MS

Zero bias

-30-20-10

010203040506070

0 1 2 3 4 5 6 7 8 9 10 11 12

GC MS VALUES (ng/ml)

Diff

eren

ce b

etw

een

met

hods

(%) GC MS VALUES Biosource values

0.11 0.150.15 0.120.17 0.190.20 0.340.25 0.240.39 0.390.61 0.511.76 1.392.27 1.773.16 2.974.70 4.247.26 5.928.01 6.439.09 8.54

11.65 9.24

“Thus, we conclude that the direct RIA method is a clinically useful assay that is appropriate for the study of the issue of “low” T within the female population”

“Reference methods to measure total T are notsuitable for routine clinical application”

Sample preparation

Zinc sulfate/methanol precipitation.

• serum/plasma samples (50 µL) in duplicate in Eppendorf tubes (1.5-mL).

• precipitating reagent (100 µL) containing D2T • vortex-mix for 1 min• centrifuged at 15 000g for 10 min• into the autosampler racks at 10 °C

ID/LC-MS/MS

• Xterra C18 column• Waters 2795 + Micromass Quattro• 40 µL injected on the column • mobile phase: water (solvent A) and

methanol (solvent B), each containingammonium acetate and formic acid

• retention time for testosterone and D2T 2.95 min and total run time 4.75 min

Analytical sensitivity

• LOD (signal-to-noise ratio >10): 0.30 nmol/l (9 ng/dL)

• Actual reference range: 15-45 ng/dL

One would prefer lower LOD

Conclusion

• low sample volume requirement• minimal sample preparation• high sample throughput• LC-MS/MS is a suitable technique for a

routine clinical biochemistry laboratoryanalyzing 8000 samples for testosteroneper year

Simultaneous determination of Androstenedione and

Testosterone by LC-MS/MS

Gallagher.L, Owen.L, Keevil.BDepartment of Clinical Biochemistry

SMUHT

Androstenedione

• C19 steroid• Produced by adrenals

(50%) and ovaries (50%)• Principle circulating

androgen in women

• Weak androgenic activity• Precursor to testosterone and estrone

Female reference range: 1.8 - 12.2nmol/L

Testosterone• C19 steroid • Produced by adrenals

(25%) ovaries (25%) and (50%) by peripheral conversion of Androstenedione

• Androgenic activity greater than Androstenedione

• Precursor for estradiol

Female reference range: <0.5 - 2.4nmol/L

Method by LCMS/MS Sample Preparation (1)

• 200μL serum, standard or QC • 10μL internal standard

(d7-A), (d2-T)• 1mL Methyl-tert-butyl-ether• Vortex mixed for 4min• Transferred supernatant into a

glass tube

Method by LCMS/MS Sample Preparation (2)

• Supernatant blown down with a dry block (40°C)

• Residue reconstituted with (100μL) 50:50 mobile phase

• Vortex mixed 1min• Placed in 96 well

microtitre plate

Liquid ChromatographyHPLC system Shimadzu

Vol injected Flow Rate

50μL 0.6mL/min

Security guard

Phenomonex C18 4x2mm cartridge column

HPLC Column Phenomonex C18 4μ, 50x3mm Synergi Hydro column

Elution Time 0 – 2min 2 – 2.5min 2.5 – 3.5min

70% Methanol (+0.1%FA+2mM AA) 95% Methanol (+0.1%FA+2mM AA) 70% Methanol (+0.1%FA+2mM AA)

FA = Formic Acid, AA = Ammonium Acetate

Mass Spectrometer

Parention

(m/z)

Production

(m/z)

Conevoltage

(V)

Collisionenergy

(eV)Androstenedione 287.3 97 26 22

d7-Androstenedione 294.4 99.9 26 26

Testosterone 289.3 109 28 28

d2-Testosterone 291.3 99 28 28

• Waters Quattro micro HPLC tandem mass spectrometer, with a Z spray ion source• ES+ ionisation mode

Tuning conditions

Mass SpectraAndrostenedione

d7-Androstenedione

Parent

Daughter

Parent

Daughter

Mass SpectraTestosterone

d2-Testosterone

Parent

Daughter

Parent

Daughter

Chromatogram

Androstenedione

Testosterone

Lower Limit of QuantificationConcentration Accuracy

(Bias%)Precision

(CV%)

0.1nmol/L 5 31.40.25nmol/L -11.5 10.90.5nmol/L -1.2 9.21.0nmol/L 1.6 3.9

Androstenedione

Concentration Accuracy(Bias%)

Precision(CV%)

0.1nmol/L -7.5 27.60.25nmol/L 7.5 11.90.5nmol/L 0.3 10.91.0nmol/L 6 6.8

Testosterone

Accuracy and Precision

Concentration Accuracy(Bias%)

Imprecision(CV%)

0.35nmol/L 14.3 6.33.5nmol/L -1.7 3.235nmol/L -2.2 4.8

Androstenedione

Testosterone

Between Batch

Concentration Accuracy(Bias%)

Imprecision(CV%)

0.3nmol/L 13 9.83.0nmol/L 9.7 3.829nmol/L 1.9 3.2

Comparison

Androstenedione:

RIA (DSL-3800 Active)

vs

LC-MS/MS

RIA vs LC- MS/MS

Bland Altman

Bias = -1.965, n = 92

Zero bias

-7

-5

-3

-1

1

0 5 10Mean of both methods

LCM

S/M

S - R

IA

0

2

4

6

8

0 5 10RIA

LC

MS/

MS

Passing Bablock

y = 0.575x - 0.207

Conclusions• A simple LC-MS/MS method for simultaneous

determination of Androstenedione and Testosterone

• This method is sensitive, accurate and precise

• Androstenedione is overestimated by RIA

• At testosterone concentrations <3nmol/L, the Roche is less accurate than LC-MS/MS

• Consider using LC-MS/MS method for all female Testosterone and Androstenedione measurements

NICHOLS INSTITUTE

“The Tradition of Innovation Continues Today”

Mass Spectrometry is emerging as the reference methodfor steroid quantitation and Nichols Institute is the firstcommercial laboratory to fully embrace this technology forthis application.

By offering superior sensitivity, specificity and diagnosticprecision, we see Mass Spectrometry emerging as the nextstandard of care for managing patients with disorders in hormone and steroid production.

Synthetic steroids

SWOT analysis• Strenghts:

– end of the black box era in Endocrinology• Weaknessess:

– investment: money AND people– technically complicated

• Opprtunities:– develop accurate and new in-house methods– revalorisation of the clinical chemist

• Threats:– quality systems/ accreditation– dangerous if expertise is lacking– few applications available from manufacturers