An Introduction to Liquid Chromatography Mass Spectrometry

Dr Kersti Karu email: [email protected] Office number: Room LG11

Recommended Textbook: “Analytical Chemistry”, G. D. Christian, P. K. Dasgupta, K.A. Schug, Wiley, 7th Edition “Trace Quantitative Analysis by Mass Spectrometry”, R.K. Boyd, C.Basic, R.A. Bethem, Wiley Applications

Lecture Overview • Rate theory - van Deemter equation for HPLC • Principle of LC analysis – HPLC subclasses – Stationary phases in HPLC – LC columns • Liquid chromatography-mass spectrometry (LC-MS) – Separation process in reversed-phase liquid chromatography – Electrospray ionisation (ESI) source – Taylor cone formation in ESI process – Quadrupole ion trap (QIT) analyser – TOF analyser – TOF analysers with V and W geometries – Orbitrap analyser – Tandem mass spectrometry • Sterolomics • Brief introduction to LC-MS practical Principle of LC-MS analysis

• HPLC: flow rate 1-2 mL/min, 4.6 mm ID column, 3-or 5-µm silica-based particles, 5-30 µL injection. Basic components of an HPLC system • Capillary LC: 200 µL/min, 1 mm ID column, 1.5- to 3-µm silica-based particles, 1-5 µL injection • Nano LC: 200 nL/min, 0.1 mm ID column, 3-µm silica-based particles, 0.1-2 µL injection • UHPLC systems (ultra-high-pressure liquid chromatography): 0.6 - 1 mL/min, 1.5- to 3- µm silica-based particles capable of pumping at 15,000 psi. Particles in LC columns

Analytes are separated based on their differential affinity between a solid stationary phase and a liquid mobile phase. The kinetics of distribution of analytes between the stationary and the mobile phase is largely diffusion-controlled. To minimise the time required for the interaction of the analytes between the mobile phase and the stationary phase two criteria should be met:- (1) the packing material should be small and as uniformly and densely packed as possible, and (2) the stationary phase should be effectively a thin uniform film with no stagnant pools and provide a small C value. Rate theory of chromatography – the van Deemter equation

The C-term in the van Deemter equation contains both mobile-phase and

stationary phase contributions (Cm and Cs)

H = A + B/푢 + Cm 푢 + Cs 푢 the Huber equation Except at very low mobile-phase velocities, the longitudinal diffusion term B is negligible because the diffusional coefficient is very small in the LC. With present uniform spherical particles of very small particle size (<5 µm) that are tightly and uniformly packed, the contribution of A term is also very small.

H = Cm 푢 + Cs 푢

The term Cs is relatively constant at a given k value; and Cm includes stagnant mobile-phase transfer (in the pores of the particles). Column efficiency is related to particle size. For well-packed HPLC columns, H is about two to three times the mean particle diameter.

H = (2 to 3) x dp

Van Deemter plots in HPLC as a function of particles size. HPLC subclasses

• Normal phase chromatography (NPC) utilises a polar stationary phase and relatively non- polar to intermediate polarity solvents such as hexane, tetrahydrofuran. • Reversed phase chromatography (RPC) utilises non-polar stationary phase C18 and polar hydroorganic solvents, ACN, MeOH, water • Hydrophilic interaction chromatography (HILIC) water is adsorbed on a hydrophilic surface to provide the partitioning process. This mode of separation suited for highly polar water soluble analytes. The basic mechanism is the same as that in NPC. ACN-water is commonly used as the eluent system, in which water is the strong eluent. • Size exclusion chromatography molecules are separated based on their size. The stationary phase is largely occupied by pores. Molecules that are larger than the largest pores cannot enter any pores and hence are “excluded” from the pores and come out in the void volume. • Ion exchange chromatography -ion exchange particles carry fixed positive or negative - + + charges, a sulfonic acid type resin, for example, has SO3 H groups where H groups can be exchange for other cations –cation exchange resin. The electrostatic interaction is governing factor in ion exchange affinities • Ion exclusion chromatography like SEC, depends on principles of exclusion to accomplish separation, all analytes elute within a finite retention window. Weak electrolytes can be separated by this technique, the dominant application area being the separation of organic acids that can be separated from strong acids and further separate according to their pKa. For

example column with a -SO3H cation exchange resin. The sulfonate group is fully ionised and the resin matrix is negatively charged. If consider a weak acid HA, the anion A- will be excluded from the interior of the resin, but no such penetration barrier exists toward a neutral molecule. The fully ionised elute in the void volume and other elute in the order of increasing pKa; acids that are largely unionised elute last. Stationary phases in HPLC

High-purity silica particles, in low trace metal content, <10µm to <2 µm diameter particles. Smaller diameter particles create higher back pressure, but they exhibit very little loss of efficiency at higher flow rates, permitting faster separation. For the pore sizes of 50 -150 A° for the analysis of small molecules; 200 - 300 A° for polypeptides and 1,000 - 4,000 A° for high molecular proteins. Most HPLC is performed in the liquid-liquid partition mode, the stationary phase is bonded to the support particles. Microporous particles- the pores being permeable to the analytes and the eluting solvent (c). Most of the mobile phase moves around the particles. The solute diffuses into the stagnant mobile phase within the pores to interact with the stationary phase, and then diffuses out into the bulk mobile phase. The use of small particles minimises the pathlenght for diffusion and hence ban broadening. • (c) Silica particles have surface silanol groups, SiOH. Silanol groups provide polar

interaction sites. The reaction with monochlorosilane R(CH3)2SiCl, where R =CH3(CH2)16-CH2- will lead to “C18 silica” stationary phase. • The extent of functionalisation is expressed in terms of wt%C (obtained by elemental analysis). • Micropores smaller then ~100A° and Macropores larger than ~1000A° and pores of intermediate size are mesopores (d). Mostly used for analysis of large molecules. • Nonporous packings (b) silica in very small particle size (1.5 µm) as there is no pores, the pore diffusion and longitudinal diffusion limitations disappear. With very small particles, the diffusion distance from the mobile to the stationary phase is short; column efficiency is virtually flow rate independent. However, the pressure needed to attain a certain flow rate thorough a given column varies inversely as the square of the particle diameter (the pressure is 1000% higher when the column is packed with 1.5 µm than with 5 µm particles). Nonporous packings have a lower surface area, limiting how much sample can be injected on column and exhibit lower RT. • (a) the packing material is composed of an inner fused or non porous particle core and a porous outer particle shell. Analytes interact with the outer shell, reducing resistance to mass transfer and providing superior separation efficiency. Particle size as small as 1.3 µm used. A monolithic column constitutes of a single solid rod that is thoroughly permeated by interconnecting pores. As with perfusion packings, they have a bimodal pore structure. Macropores, which act as flow-through pores, are about 2 µm in diameter. The silica skeleton contains mesopores with diameters of about 130 A°. It can be surface modified with stationary phase like C18. The rod is shrink-wrapped in a polyetheretherketone (PEEK) plastic holder. The surface area of the mesopores is about 300 m2/g, and the total porosity is 80%. The column exhibits a van Deemter curve close to that for 3.5-µm packed particles, but with a pressure drop ~40% of the packed column run at the same flow rate. Polymeric monoliths are available in capillary formats from 0.1 to 1 mm in diameter and up to 250 mm in length. Stationary phases for hydrophilic interaction chromatography (HILIC) three types of HILIC phases:- neutral, charged and zwitterionic. The neutral HILIC phase contains amide or diol functionalities bonded to porous silica. A charged phase exhibits strong electrostatic interactions and may consist of bare silica or amino, aminoalkyl or sulfonate functionalities bonded to porous silica. The separation selectivity between different analytes can be favoured by the electrostatic interactions that are contributing to the retention with the charged HILIC stationary phases. A zwitterionic bonded phase, - + a O3S(CH2)3-N (CH3)2CH2- zwitterionic group is the functional entity bonded to a porous silica support, or a polymeric support for greater pH range. Liquid chromatography – mass spectrometry (LC-MS)

Sample injection system

LC Columns Separation process in reversed-phase liquid chromatography

S1 S1 S2

A reversed phase LC column contains porous particles coated with an organic stationary phase, e.g., a C18 hydrocarbon chain. The mobile phase carries analytes around and through the particles. The order of elution is determined by the length of time individual analytes partitioned in the stationary phase. Compounds elute in reverse order of their polarity, the most polar compounds elute earlier. Electrospray ionisation (ESI) source

analyser

Two concentric steel tubes carry the liquid and nebulising gas. After nebulisation, additional gas is used to dry the droplets. As the droplets shrink the charge density increases to the point at which the droplets are no longer stable and ions are released from the liquid matrix into the vapour phase. Ions enter the mass analyser at an angle to reduce the entrainment of neutral molecules. At low flow rates, such as in nanospray ESI, spontaneous evaporation is sufficient to eliminate the need for the drying gas. Taylor cone formation occurs both at the end of the ESI tube and as the droplets disintegrate to release ions.

Stable nebulisation of the liquid flow occurs when a Taylor cone is established at the tip of the ESI source. As the droplets are dried the charge density increases resulting in droplet disintegration either because of Coulombic forces or by distortion to form a Taylor cone from which ions are released. Quadrupole ion trap (QIT) analyser

electrode Ions injected into the trap are held by rf and dc voltages placed on the hyperbolic ring electrode and end caps. Ions move in a complex sinusoidal path where there are crossover points. Space-charge effects occur at the crossover points of the ion paths when too many ions are present. By changing the voltages progressively ions with different m/z values are ejected sequentially onto the detector, generating spectra. MSMS fragmentation takes place when a specific m/z is isolated in the trap and excited in the presence of a neutral gas, providing MS/MS data. The isolation/fragmentation cycle can be repeated to obtain MSn data. Injection of ions into an orthogonal TOF analyser

The basic principle of TOF analyser:- pulses of ions are accelerated from the ion source into the analyser tube, and the time for an ion to travel through a field-free region to the detector is measured. The time-of-flight for the passage of an ion in a TOF analyser is a function of momentum, and therefore its m/z. The acceleration voltage and (consequently) the kinetic energy (momentum), is the same for all ions. Thus those with the lowest m/z will travel fastest and arrive at the detector first, followed by the sequential arrival of ions with successively higher m/z. API focusing pusher ion source region (pulsed voltage) part of ion beam

API atmospheric direction of ions in the analyser is pressure ionisation composed of horizontal and vertical components imparted by the source and pusher voltages. Ions are generated continuously in API sources, thus, the individual packets of ions required in TOF analysis must be excised from the ion beam, and accelerated orthogonally into the analyser using a pulsed pusher voltage. Only part of the continuous ion beam can be sampled as the excised ion packet must traverses the analyser before another set of ions can be introduced. The duty cycle of the orthogonal injection process limits the sensitivity of orthogonal TOF instruments. Principle of orthogonal TOF analysers with V and W geometries

secondary ion mirror pusher

Ion path

V geometry W geometry

Ions arriving from the source are accelerated orthogonally into the analyser by a pulsed voltage from the pusher. Ions separate based on their momenta. As they travel through the analyser the lightest, fastest, ions (lowest m/z) will arrive at the detector first. Some horizontal momentum imparted in the source remains so that ions travel at an angle into and out of the reflectron thus attaining a characteristic ‘V’ trajectory. Increasing the distance that the ions travel improves mass resolution, e.g., by using ‘W’ geometry. Orbitrap analyser induced transient ion input is off center

and oscillate along, the specially shaped inner electrode creating a complex ion path.

Ions, typically from ESI and APCI, are collected in a specialised component called the C-trap and them injected into the orbitrap as high-speed pulses. Ions are injected at an angle and offset from the centre of the trap. The momentum of the ions causes them to orbit around, and oscillate along, the central spindle-like electrode. The lateral oscillation of the ions along the inner electrode induces a transient (image) current in the split outer electrode. The recorded image current is interpreted using Fourier transform analysis to provide m/z values and intensities. Principle of a QTOF MS/MS instrument

API CID

Quadrupoles as ion guide (q) the rf voltages can be applied to the quadrupole, but without being combined with a dc field. Under these conditions all ions, e.g. those emerging from the ion source that enter the q are transmitted, regardless of their m/z values. Ions are produced in an atmospheric pressure ionisation (API) source. The quadrupole can be used in broad bandpass (q) mode, to pass all ions to the time-of-flight (TOF) analyser, or in narrow band pass (Q) mode to select an ion with a specific m/z for collision induced dissociation (CID). The CID cell also has a q function by which the fragments formed are constrained and transferred to the TOF analyser. CID fragment ions are separated and collected in the TOF analyser. Schematic of a hybrid linear ion trap with an orbitrap or ion cyclotron resonance (ICR) cell as the second analyzer second

The LIT-orbitrap combination can be used for both MS and MSn experiments because the LIT has its own detector. Additional fragmentation at a different collision energy can be undertaken in a second collision cell. Products from either collision cell are injected, via the C trap, into the orbitrap where transients are collected for subsequent FT analysis.

(b) ICR cell superconducting magnet In the LIT-ICRMS, ions from the LIT traverse a set of extended optics into the ICR cell located within a superconducting magnet. A ramped rf voltage pulse is used to coalesce different m/z values and move them close to the receiver plates where transients are collected for subsequent FT analysis. High resolution improves accurate mass measurement STEROIDS

Cyclopentanoperhydrophenanthrene skeleton

Natural steroids have 5α-Cholestane structure with two methyls IUPAC numbering showing four fused-ring isoprene structure Steroids in Brain

• 25% body’s cholesterol is present in brain • Cholesterol formed de novo • 1st step of cholesterol metabolism formation of an oxysterol

OH HO 24S-Hydroxycholesterol

Cholesterol

Lütjohann Acta Neurol Scand 2006 114 33 Oxysterols in brain

OH OH

(S)

(R) (R)

(R) (R) (S) (S) HO (Z) HO (Z) ng/mg ng-pg/mg

OH

(R)

(R) (S)

(R) (R) (S) (S) (S) HO (Z) OH HO (Z) • Transport forms of cholesterol • Oxysterols are ligands for LXR • Oxysterols implicated in neurodegenerative disease (AD)

• Oxysterols formed by reaction with O3 or ROS as a result of inflammation

OH HO

Björkhem et al Curr Opin Lipidol 2002 13 247 Analytical Strategy

st nd • Extraction: 1 C2H5OH, 2 CH3OH/CH2Cl2 • Separation: Straight phase • Derivatisation: • LC-ES-MSn

OH HO •Specificity for 3b-OH •Enhance Solubility •Enhance Sensitivity •x 100

Smith & Brooks J Chromatogr 1974 101 373 Wang et al Anal Chem 2006 78 164 nano-ESI Mass Spectrum C5-3β,24S-diol C4-24S-ol-3-one GP hydrazone OH [M]+ Ion Current: 8x107 100 534

[M]+ N 80 m/z 534 HN + N O

60

OH Relative Abundance, % Abundance, Relative

40

[M+H]+ HO m/z 403 20 + [M+H-H2O] + [M+H-2xH2O] 385 367

0 360 400 440 480 520 m/z Oxysterols in rat brain

100 mg brain Inject ~ 0.1 mg cholesterol

518 100 hydroxycholesterol

534

dihydroxycholesterol % dehydrocholesterol

333 550

373 665

0 m/z 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 + 2 C4-24S-ol-3-one GP hydrazone m/z 534 [M] MS

OH OH 100 455 [M-79]+

+ [M] [M-79]+ N m/z 534 N m/z 455 HN N HN O O

OH

+ +

[M-79-H20] [M-79-CO] Relative Abundance, % Abundance, Relative N m/z 437 N m/z 427 NH + HN [M-79-H2O] O [M-79-CO]+

427

437 163 231 325 353 394 409 150 200 250 300 350 400 450 m/z m/z 534 [M]+m/z 455 [M-79]+

C4-24S-ol-3-one 3 OH + MS [M-79-H2O] '*f-H GP hydrazone 437 HN -H *b -C H '*e HN 3 2 4 m/z 163 O *b3-C2H4 N HN *b2 *b1-12 HN CH2 O HN CH3 m/z 455 *b2 [M-79]+ O *b3-C2H4 m/z 177 163 [M-107]+

N Relative Abundance, % Abundance, Relative *b -12 HN *b1-12 427 1 m/z 151 151 ‘*f O 353

409

*b2 ‘*e 394 177 325 367 135 231

150 200 250 300 350 400 m/z Ion Source Ions Ions Signal Cap-LC Mass Analyser Detector Computer Nano-ES Ion trap Vacuum Chromatography

• LC-MSn • Ultimate 3000 HPLC – LTQduo ion trap MS

• PepMap C18 Column (3 µm particles, 180 µm x 150 mm) • 800 mL/min • (A) 50% MeOH, 0.1% FA • (B) 95% MeOH, 0.1% FA • 30%(B)  80%(B) (80%)  30%(B) 15min 10min 10min 25.8 capLC - MS3 N OH HN 4 N+ C -7ß-ol-3-one GP N C4 -ol-3-one GP O 22.2 HN N OH O

(S) 21.9 23.1

4 N C -22S-ol-3-one GP 19.4 HN N O OH 19.6 O (S) 19.1 N 4 HN C -7-one-3-one GP N O

C4 -24S-ol-3-one GP N 18.8 HN N O OH

OH 16.5 15.4 C4 -25-ol-3-one GP N HN C4 -7a,27-diol-3-one GP N 11.7 O N OH 100 HN N+ O 60 O C OH 20 N BA4 -3-one GP HN 0 20 40 N Time (min) O Chromatography

• LC-MSn • Surveyor HPLC – LTQ-Orbitrap • Hypersil GOLD Column (1.9 mm particles, 50 x 2.1 mm) • 200 mL/min • (A) 50% MeOH, 0.1% FA • (B) 95% MeOH, 0.1% FA • 20%(B)  80%(B) (80%)  20%(B) 7min 5min 4min LC-MSn Strategy (m/z 300 - 600) ESI_Unisil2_070206_fr1 03/05/2006 18:23:26

RT: 0.00 - 17.00 12.53 NL: 7.89E5 100 m/z= 518.4079-518.4131 F: FTMS 12.38 + p ESI Full ms [ 320.00-600.00] 50 MS ESI_Unisil2_070206_fr1 0.48 2.11 5.17 6.12 6.95 7.79 8.19 9.18 10.39 11.05 11.73 12.91 13.65 14.62 15.62 0 7.60 NL: 8.91E5 100 m/z= 532.3871-532.3925 F: FTMS + p ESI Full ms [ 320.00-600.00] 50 MS ESI_Unisil2_070206_fr1 6.85 10.23 7.81 9.79 10.42 0 7.35 NL: 5.29E7 100 m/z= 534.4027-534.4081 F: FTMS 7.65 + p ESI Full ms [ 320.00-600.00] 50 MS ESI_Unisil2_070206_fr1 7.87 9.48 10.03 10.87 12.38 12.90 14.57 0 8.88 NL: 1.95E4 100 9.05 m/z= 546.3663-546.3717 F: FTMS + p ESI Full ms [ 320.00-600.00] 8.34 8.48 9.23 50 MS ESI_Unisil2_070206_fr1 0 5.49 NL: 2.16E5 100 m/z= 548.3820-548.3874 F: FTMS + p ESI Full ms [ 320.00-600.00] 50 6.17 6.26 8.99 MS ESI_Unisil2_070206_fr1 7.22 9.37 9.96 10.62 0 5.45 NL: 3.30E5 100 4.47 m/z= 550.3975-550.4031 F: FTMS + p ESI Full ms [ 320.00-600.00] 50 5.06 4.31 5.91 9.20 MS ESI_Unisil2_070206_fr1 6.45 8.76 9.43 0 10.03 NL: 6.34E5 100 m/z= 552.4132-552.4188 F: FTMS + p ESI Full ms [ 320.00-600.00] 50 9.23 MS ESI_Unisil2_070206_fr1 0 5.43 NL: 7.62E4 100 6.15 m/z= 564.3768-564.3824 F: FTMS 8.37 + p ESI Full ms [ 320.00-600.00] 50 6.59 MS ESI_Unisil2_070206_fr1 7.76 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Time (min) Data From LTQ-Orbitrap (Exact Mass RIC ±5ppm) Reconstructed ion chromatogram (RIC) fro m/z 534 a 7.35 RT: 0.00 - 17.00 100 NL: 5.29E7 m/z= 95 534.4027-534.4081 F: 90 FTMS + p ESI Full ms [ 85 320.00-600.00] MS ESI_Unisil2_070206_fr 80 1 75 70 OH OH 65 (S) (S) 60 7.65 55 50 O N 45 HN (Z) N (E) 40 (Z) N (Z) C H N O + 35 34 52 3 2 HN Exact Mass: 534.4054 C N 30 O 25 C H N O + 20 34 52 3 2 Exact Mass: 534.40540 15 10 5 0 0 2 4 6 8 10 12 14 16 534MS2

24S_OH_C #51 RT: 5.45 AV: 1 NL: 5.99E6 M-79 F: FTMS + p ESI Full ms2 [email protected] [ 145.00-550.00] 455.3630 100

OH OH 90

80

70

60 N N HN HN C C N 50 O O + C29H47N2O2 C H N O + 40 Exact Mass: 455.36320 34 52 3 2

RelativeAbundance Exact Mass: 534.40540

30

20 M-107

10 427.3685

0 150 200 250 300 350 400 450 500 550 m/z 534455MS3 24(S)-hydroxycholesterol M-79-18 24S_OH_C #67-80 RT: 6.36-7.10 AV: 14 NL: 2.03E6 F: FTMS + p ESI Full ms3 [email protected] [email protected] [ 125.00-550.00] 437.3528 100

'*f OH 90

H 80

70

N *b -28 60 N N 3 + *b -12 *b2 HN HN C29H45N2O HN 1 + C C C23H33N2O C Exact Mass: 437.35264 50 + O Exact Mass: 353.25874 O O C29H47N2O2 Exact Mass: 455.36320 40

RelativeAbundance

30 *b3-28 M-107

163.0865 20 ‘*f 427.3686 *b -12 1 409.3580 151.0865 353.2591 10 193.2107 *b2 394.3471

0 150 200 250 300 350 400 450 500 550 m/z The prenatal diagnosis of Smith-Lemli-Opitz syndrome from amniotic fluid

Squalene

O Squalene Epoxidase (EC 1.14.99.7)

Squalene Epoxidase (EC 1.14.99.7) 2,3-Oxidosqualene 2,3:22,23-Dioxidosqualene O O H O H H (EC 5.4.99.7) Lanosterol synthase/OSC/ H 2,3-Epoxysqualene- H H Lanosterol Cyclase (EC 5.4.99.7)

3b-Hydroxysterol 24-reductase/ Desmosterol reductase HO HO (EC:1.3.1.72) HO H H H 24S,25-Epoxylanosterol Lanosterol Dihydrolanosterol H O CYP51A/ H H (EC 1.14.13.70) (EC 1.14.13.70) Lanosterol-14--demethylase H (EC 1.14.13.70) H H

(EC:1.3.1.72)

HO HO HO H H H 4,4-Dimethylcholesta-8(9),14,24-trien-3b-ol 4,4-Dimethylcholesta-8(9),14-dien-3b-ol

(EC 1.3.1.70) H O H H Sterol C14-reductase/ (EC 1.3.1.70) 14-Sterol reductase/ H H H 3b-Hydroxysterol-14-reductase (EC 1.3.1.70)

HO HO (EC:1.3.1.72) HO H H H 4,4-Dimethylcholesta-8(9),24-dien-3b-ol C-4 Methylsterol oxidase/ 4,4-Dimethylcholesta-8(9)-en-3b-ol Methylsterol monoxygenase (EC 1.14.13.72) H O (EC 1.14.13.72) H (EC 1.14.13.72) H (EC 1.1.1.170) C4-Sterol decarboxylase/ (EC 1.1.1.170) H Sterol-4-carboxylate 3-dehydrogenase H H (decarboxylating) (EC 1.1.1.170)

HO HO HO H H H Zymosterol (EC:1.3.1.72) Cholesta-8(9)-en-3b-ol 3b-Hydroxysterol 8,7-isomerase H O (EC 5.3.3.5) H H (EC 5.3.3.5) (EC 5.3.3.5)

H H H

(EC:1.3.1.72) HO HO HO H H H Cholesta-7,24-dien-3b-ol Lathosterol/ Cholesta-7-en-3b-ol H O Lathosterol 5-desaturase/ H (EC 1.14.21.6) Sterol-C5-desaturase (EC 1.14.21.6) H (EC 1.14.21.6) H H H

(EC:1.3.1.72)

HO HO HO 7-Dehydrodesmosterol 7-Dehydrocholesterol H O H H (EC 1.3.1.21) (EC 1.3.1.21) Sterol 7-reductase/ H 3b-Hydroxysterol 7-reductase H H (EC 1.3.1.21)

(EC:1.3.1.72) Cholesterol HO 24S,25-Epoxycholesterol HO Desmosterol HO Sterol analysis for the prenatal diagnosis of Smith-Lemli-Opitz syndrome 25.83 100 Cholesterol %RA 518→439

100 24.46 Desmosterol %RA 516→437

100 23.90 7-DHC %RA 516→437

0 10 20 30 40 Time (min) 0.002-0.01 µL amniotic fluid injected on-column

(a) (b) 25.83 25.83 100 100 Ion Count 6.7E6 Ion Count 5.58E6 %RA %RA MS2 : 518→439 MS2 : 518→439

23.90 100 100 23.90 Ion Count 9.85E4 %RA %RA Ion Count 6.27E6 MS2 : 516→437 MS2 : 516→437

0 10 20 30 40 0 10 20 30 40 Time (min) Time (min) 1.20

1.00

0.80

0.60

0.40

0.20

0.00

31576 30488 30355 31414 32288 33952 34048 34107 34105 34070 34106 34089 34104 30079 32688 34090 33953 29986

Mean ratio of 7/8-DHC to cholesterol for 18 amniotic fluids from SLOS- affected and non-affected pregnancies. Eighteen samples were subjected to oxidation/GP-derivatisation reactions in triplicate and were analysed by cap- LC-MSn. Phytosterol practical

Cholesterol β-sitosterol Stigmasterol 5-cholesten-3β-ol 5-cholesten-24β-ethyl-3β-ol 5,22-cholestadien-24β-ethyl-3β-ol

Campesterol Stigmastanol 5-cholesten-24α-methyl-3β-ol 5-cholestan-24β-ethyl-3β-ol Analytical Strategy

• Extraction of phytosterols : C2H5OH • Derivatisation with GP hydrazine • Direct infusion ESI onto the QTOF • LC-ESI-MSn Direct infusion of a sample into the Liquid chromatography - Mass mass spectrometer Spectrometry analysis

capLC-MS: Separate components of interest, giving sharp peaks (10 s – 1 min) away from the solvent front