Kit and method for quantitative detection of steroids

Abstract

The invention relates to a kit and methods for quantitative detection of steroids in a sample. The kit comprises quantitative charge tags and an oxidizing agent.

Claims

1. A method for the quantitative detection of oxosteroids and hydroxy steroids in a sample, the method comprising: i. reacting a first portion of the sample with a first member of a pair of quantitative charge tags of formula (I) or formula (II); wherein R is hydrogen, C.sub.1-4 alkyl or aryl, OR.sup.1 or NR.sup.1R.sup.2, each R.sup.1 and R.sup.2 is C.sub.1-3 alkyl or R.sup.1 and R.sup.2 together with the nitrogen atom to which they are attached form a 5- or 6-membered heteroalkyl ring; each R.sup.3 is independently C.sub.1-3 alkyl; and X is a halide ion; ii. reacting a second portion of the sample with an agent capable of oxidising an OH group to a carbonyl group; iii. reacting the product of step (ii) with a second member of a pair of quantitative charge tags of formula (I) or formula (II); iv. combining the products of steps (i) and (iii); v. conducting mass spectrometry on the combined product of step (iv) and determining the quantities therein of compounds labelled with the first and second members of the pair of quantitative charge tags, thereby providing quantitative detection of both oxosteroids and hydroxy steroids in a single analysis.

2. The method as claimed in claim 1, wherein the first and second portions of the sample are of equal volume.

3. The method as claimed in claim 1 wherein the quantitative charge tags are differential mass tags and the mass spectrometry of step (v) is liquid chromatography-mass spectrometry (LC-MS) or HPLC-MS.

4. The method as claimed in claim 1 wherein the quantitative charge tags are isobaric mass tags and the mass spectrometry of step (v) is an MS/MS method.

5. The method as claimed in claim 1 wherein the sample is prepared from a body fluid, for example whole blood, plasma, serum, cerebrospinal fluid, sputum, tears, sweat or urine; or from a specimen of tissue, hair, nails by extraction of the steroids into a solvent.

6. A method for the quantitative detection of oxosteroids and hydroxy steroids in a sample, the method comprising: i. reacting the sample with a first member of a pair of quantitative charge tags of formula (I) or formula (II) wherein R is hydrogen, C.sub.1-4 alkyl or aryl, OR.sup.1 or NR.sup.1OR.sup.2; each R.sup.1 and R.sup.2 is C.sub.1-3 alkyl or R.sup.1 and R.sup.2 together with the nitrogen atom to which they are attached form a 5- or 6-membered heteroalkyl ring; each R.sup.3 is independently C.sub.1-3 alkyl; and X is a halide ion; ii. reacting a reference composition with a second member of a pair of quantitative charge tags of formula (I) or formula (II); iii. combining the products of steps (i) and (ii); iv. conducting mass spectrometry on the combined product of step (iii) and determining the quantities therein of compounds labelled with the first and second members of the pair of quantitative charge tags, thereby providing quantitative detection of both oxosteroids and hydroxy steroids in a single analysis.

7. The method according to claim 6 wherein both the sample and the reference composition are reacted with an agent capable of oxidising an OH group to a carbonyl group before reaction with the quantitative charge tags.

8. The method as claimed in claim 6, wherein the sample and the reference composition are of equal volume.

9. The method as claimed in claim 6 wherein the quantitative charge tags are differential mass tags and the mass spectrometry of step (vi) is liquid chromatography-mass spectrometry (LC-MS) or HPLC-MS.

10. The method as claimed in claim 6 wherein the quantitative charge tags are isobaric mass tags and the mass spectrometry of step (vi) is an MS/MS method.

11. The method as claimed in claim 6 wherein the method is repeated for a further sample using the same or a different reference composition.

12. A method for the quantitative determination of oxosteroids and hydroxy steroids in a sample comprising: i. reacting a first portion of the sample with a first member of a first pair of quantitative charge tags of formula (I) or formula (II); wherein R is hydrogen, C.sub.1-4 alkyl or aryl, OR.sup.1 or NR.sup.1R.sup.2; each R.sup.1 and R.sup.2 is C.sub.1-3 alkyl or R.sup.1 and R.sup.2 together with the nitrogen atom to which they are attached form a 5- or 6-membered heteroalkyl ring; each R.sup.3 is independently C.sub.1-3 alkyl; and X is a halide ion; ia. reacting a first portion of a reference composition with a first member of a second pair of quantitative charge tags of formula (I) or formula (II); ii. reacting a second portion of the sample with an agent capable of oxidising an OH group to a carbonyl group; iia. reacting a second portion of a reference composition with an agent capable of oxidising an OH group to a carbonyl group; iii. reacting the product of step (ii) with a second member of a first pair of quantitative charge tags of formula (I) or formula (II); iiia. reacting the product of step (iia) with a second member of a second pair of quantitative charge tags of formula (I) or formula (II); iv. combining the products of steps (i) and (iii); v. conducting mass spectrometry on the combined product of step (iv) and determining the quantities therein of compounds labelled with the first and second members of the pair of quantitative charge tags, thereby providing quantitative detection of both oxosteroids and hydroxy steroids.

13. The method as claimed in claim 12 wherein the portions of sample and reference composition are of equal volume.

14. The method as claimed in claim 12 wherein the first pair of quantitative charge tags are differential mass tags and the second pair of quantitative charge tags are isobaric mass tags.

15. The method as claimed in claim 12 wherein the first pair of quantitative charge tags are isobaric mass tags and the second pair of quantitative charge tags are differential mass tags.

16. The method as claimed in claim 12 further comprising repeating steps (i) to (iii) with a further sample and a further pair of quantitative mass tags and combining the labelled further sample portions with the product of step (iv) before carrying out mass spectrometry.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention will now be described in greater detail with reference to the examples and to the drawings in which:

(2) FIG. 1A is a schematic illustration of the design and application of quantitative charge-tags using differential mass tags.

(3) FIG. 1B is a schematic illustration of the design and application of quantitative charge-tags using isobaric mass tags, wherein [M-Py].sup.+ corresponds to the loss of pyridine from the molecular ion [M].sup.+.

(4) FIG. 1C shows use of quantitative charge-tags to deconvolute steroids oxidised to contain a 3-oxo group from those naturally possessing an oxo group, wherein CHO represents enzymatic oxidation with cholesterol oxidase.

(5) FIG. 1D shows relative quantification of steroids in target (e.g. AD) plasma against control.

(6) FIG. 1E shows quadruplex use of quantitative charge-tags, wherein plasma samples (e.g. patient and control) are analysed with and without enzymatic oxidation.

(7) FIG. 2A illustrates the validation of quantitative charge-tags using ESI-MS recorded on a Q-TOF Ultima showing the peak intensity ratio for a 1:1 mixtures of cholesterol (Chol) and 25-hydroxycholesterol (25-HC) labelled with [.sup.2H.sub.0]-GP (i) and [.sup.2H.sub.5]-GP (ii).

(8) FIG. 2B illustrates the validation of quantitative charge-tags using LC-MS reconstructed-ion chromatograms (RICs) for 3β-hydroxycholest-5-enoic acid (HCA) in a 1:1 mixture of a plasma sample labelled with [.sup.2H.sub.0]-GP (i) and [.sup.2H.sub.5]-GP (ii), wherein the mass spectrum at the peak apex of 7.38 min is shown in the lower panel, and the measured ratio after normalisation with [.sup.2H.sub.6]24(R/S)hydroxycholesterol internal standard is shown in (C).

(9) FIG. 2C illustrates the validation of quantitative charge-tags using LC-MS RIC peak intensity ratios for 3β-hydroxycholest-5-enoic acid (548/553) and 3β,7α-dihydroxycholest-5-enoic plus 7α-hydroxy-3-oxocholest-5-enoic acids (564/569) in mixtures of plasma samples labelled with [.sup.2H.sub.0]-GP (i) and [.sup.2H.sub.5]-GP (ii).

(10) FIG. 3A shows an LC-MS analysis of a plasma sample from a child with a neurological disease of unknown aetiology, with RIC of m/z 553.4155 corresponding to [M].sup.+ ions of 3β-hydroxycholest-5-enoic (HCA) and 3-oxocholest-4-enoic (CAO) acids derivatised with [.sup.2H.sub.5]-GP (ii) following cholesterol oxidase treatment, and of m/z 548.3847 corresponding to [M].sup.+ ions of 3-oxocholest-4-enoic acid derivatised with [.sup.2H.sub.0]-GP (i) in the absence of cholesterol oxidase treatment. The chromatograms are plotted on an identical intensity scale. The absence of an appropriate peak in the RIC of m/z 548.3847 indicates that no 3-oxocholest-4-enoic acid is present in plasma. The lower panel shows the mass spectrum recorded at the peak apex of 7.09 min.

(11) FIG. 3B shows an LC-MS analysis of a plasma sample from a child with a neurological disease of unknown aetiology, with RIC of m/z 555.4312 corresponding to [M].sup.+ ions of dihydroxycholesterols (diHC) and dihydroxycholestenones (diHCO) derivatised with [.sup.2H.sub.5]-GP (ii) following cholesterol oxidase treatment, and of m/z 550.4003 corresponding to [M].sup.+ ions of dihydroxycholestenones derivatised with [.sup.2H.sub.0]-GP (i) in the absence of cholesterol oxidase treatment. The presence of appropriate peaks in the RIC of m/z 550.4003 indicates that dihydroxycholestenones are present in the sample. The enhanced abundance of peaks in the RIC of m/z 555.4312 over m/z 550.4003 indicates that dihydroxycholesterols are also present. The chromatograms are plotted on an identical intensity scale. The lower panel shows the mass spectrum recorded at the peak apex of 5.51 min. Both the 7α,25- and 7α,26-dihydroycholesterol/dihydroxycholesten-3-one isomers appear as syn and anti conformers following derivatisation.

(12) FIG. 3C shows an LC-MS analysis of a plasma sample from a child with a neurological disease of unknown aetiology, with RIC of m/z 569.4105 corresponding to [M].sup.+ ions of dihydroxycholestenoic (diHCA) and hydroxyoxocholestenoic (HCAO) acids derivatised with [.sup.2H.sub.5]-GP (ii) following cholesterol oxidase treatment, and of m/z 564.3796 corresponding to [M].sup.+ ions of hydroxyoxocholestenoic acids derivatised with [.sup.2H.sub.0]-GP (i) in the absence of cholesterol oxidase treatment. The chromatograms are plotted on an identical intensity scale. The lower panel shows the mass spectrum recorded at the peak apex of 5.19 min. Both the 3β,7α-dihydroycholest-5-enoic/7α-hydroxy-3-oxocholesten-4-enoic acids appear as syn and anti conformers following derivatisation.

(13) FIG. 4A depicts characterisation of inborn errors of metabolism from patient plasma, with RIC of 539.4363 and 534.4054 corresponding to [M].sup.+ ions of monohydroxycholesterols (HC) labelled with [.sup.2H.sub.5]-GP (ii) from patient plasma and with [.sup.2H.sub.0]-GP (i) from control plasma. Both chromatograms are plotted on an identical intensity scale. The lower panels are mass spectra recorded at peak apex of 7.47 min and 7.16 min, respectively. The chromatograms define the inborn error of metabolism to be oxysterol 7α-hydroxylase deficiency.

(14) FIG. 4B depicts characterisation of inborn errors of metabolism from patient plasma, with RIC of 553.4155 and 548.3847 corresponding to [M].sup.+ ions of 3β-hydroxycholest-5-enoic acid (HCA) labelled with [.sup.2H.sub.5]-GP (ii) from patient plasma and with [.sup.2H.sub.0]-GP (i) from control plasma. Both chromatograms are plotted on an identical intensity scale. The lower panels are mass spectra recorded at peak apex of 7.47 min and 7.16 min, respectively. The chromatograms define the inborn error of metabolism to be oxysterol 7α-hydroxylase deficiency.

(15) FIG. 4C depicts characterisation of inborn errors of metabolism from patient plasma, with RIC of m/z 539.4363 and 534.4054 corresponding to [M].sup.+ ions of monohydroxycholesterols and hydroxycholest-4-en-3-ones (HCO) labelled with [.sup.2H.sub.5]-GP (ii) from a different patient plasma and with [.sup.2H.sub.0]-GP (i) from control plasma. Both chromatograms are plotted on an identical intensity scale. The lower panel shows the mass spectrum at the peak apex of 9.75 min. The chromatograms define the inborn error of metabolism to be CTX.

(16) FIG. 4D depicts characterisation of inborn errors of metabolism from patient plasma, with MS.sup.2 RIC of m/z 457.3699 and 456.3666 corresponding to [M-Py].sup.+ ions of monohydroxycholesterols and hydroxycholestenones labelled with [.sup.13C.sub.2]-GP (iii) from patient plasma and with [.sup.13C.sup.15N]-GP (iv) from control plasma. Both chromatograms are plotted on an identical intensity scale. The lower panel shows the MS.sup.2 spectrum acquired at the peak apex of 9.79 min. The chromatograms define the inborn error of metabolism to be CTX.

(17) FIG. 4E depicts characterisation of inborn errors of metabolism from patient plasma, with MS.sup.3 total ion chromatogram (TIC) of monohydroxycholesterols and hydroxycholest-4-en-3-ones labelled with [.sup.13C.sub.2]-GP (iii) from patient plasma and with [.sup.13C.sup.15N]-GP (iv) from control plasma. Both chromatograms are plotted on an identical intensity scale. The lower panel shows the MS.sup.3 spectrum recorded at the peak apex 9.80 min. The chromatograms define the inborn error of metabolism to be CTX.

(18) FIG. 4F depicts characterisation of inborn errors of metabolism from patient plasma, with RIC of m/z 555.4312 and 550.4003 corresponding to [M].sup.+ ions of dihydroxycholesterol (diHC) and dihydroxyoxocholesterol (diHCO) labelled with [.sup.2H.sub.5]-GP (ii) from patient plasma and with [.sup.2H.sub.0]-GP (i) from control plasma. Both chromatograms are plotted on an identical intensity scale. The lower panel shows the mass spectrum recorded at the peak apex of 8.45 min. The chromatograms define the inborn error of metabolism to be CTX.

(19) FIG. 5A shows RIC of 521.4257 and 516.3948 corresponding to [M].sup.+ ions of dehydrocholesterols (dHC) labelled with [.sup.2H.sub.5]-GP (ii) from patient plasma and with [.sup.2H.sub.0]-GP (i) from control plasma. Both chromatograms are plotted on an identical intensity scale. The lower panel shows the mass spectrum recorded at the peak apex of 11.96 min.

(20) FIG. 5B shows MS.sup.3 TIC of dehydrocholesterols labelled with [.sup.2H.sub.5]-GP (ii) from patient plasma and with [.sup.2H.sub.0]-GP (i) from control plasma. Both chromatograms are plotted on an identical intensity scale. Data was obtained by on the LTQ-Orbitrap. The lower panel shows the MS.sup.3 spectrum recorded at the peak apex of 11.94 min.

(21) FIG. 6 illustrates the validation of isobaric mass tags using a similar method to that used for FIG. 2 and a 1:10 ratio of cholesterol (Chol) and 25-hydroxycholesterol (25-HC) labelled with [.sup.2H.sub.0]-GP (i of Table 1) and [.sup.2H.sub.5]-GP (ii of Table 1).

(22) FIG. 7A shows analysis of pooled samples of plasma from controls, patients with Alzheimer's disease (AD) and mild cognitive impairment (MCI) and demonstrates that isomers of androstanolone 3-sulphate show differential abundance in the three samples.

(23) FIG. 7B shows that levels of isomers of androstanolone 3-sulphate fall from control to MCI to AD patients.

(24) FIG. 8 shows a scheme for the extraction of steroids from plasma.

DETAILED DESCRIPTION

EXAMPLES

(25) Material

(26) Authentic sterols were purchased from Avanti Polar Lipids (Alabaster, Ala.) or Sigma Aldrich and stored at −20° C. until use. HPLC water and HPLC grade solvents were purchased from Fisher Scientific. GP reagent (chloride salt) was purchased from TCI Europe (Oxford, UK). Cholesterol oxidase from Streptomyces sp. was from Sigma Aldrich. Certified Sep-Pak C.sub.18 cartridges were from Waters (Elstree UK). All other chemicals and reagents were purchased from Sigma Aldrich or Fisher Scientific and used as received unless otherwise stated. Blood plasma samples were from St Mary's Hospital, Manchester, Institute of Child Health, London or from a GlaxoSmithKline study and were provided with institutional review board and ethical approval.

Example 1—Synthesis of Quantitative Charge-Tags

(27) The isotope-labelled quantitative charge-tags were synthesized from pyridine, ethyl bromoacetate and hydrazine as illustrated in Scheme 1 with high yields (97%).

(28) ##STR00024##

(29) To a solution of pyridine (1.0 mL, 11.6 mmol) in ethanol (10 mL), ethyl bromoacetate (1.51 mL, 11.6 mmol) was added dropwise. The resulting mixture was heated at reflux for 4 hr then allowed to cool to room temperature then to 0° C. Hydrazine hydrate (80% aqueous solution, 0.73 mL, 11.6 mmol) was added carefully causing a white precipitate to form. The precipitate was recovered by vacuum filtration and dried under reduced pressure to afford the GP reagent (i of Table 1) as a white solid (2.66 g, 11.2 mmol, 97%). Isotope labelled versions of the GP reagent are synthesised in an identical manner but using [.sup.2H.sub.5]pyridine, ethyl [1,2-.sup.13C.sub.2]bromoacetate, [.sup.15N]pyridine, ethyl [1-.sup.13C]bromoacetate, and [.sup.2H.sub.5.sup.15N]pyridine (see Table 1).

(30) Initial synthetic procedures were performed with [.sup.2H.sub.5]pyridine of 99.5% isotopic purity, this led to 0.5% of the ultimate product (ii of Table 1) being unlabelled. When patient samples were labelled with the GP reagent (ii of Table 1), the presence of unlabelled GP reagent in the reaction mix distorted the signal for metabolites labelled with GP reagent (i of Table 1) from e.g. control samples, when analysed in the same LC-MS run (FIG. 1D). In most cases this distortion was insignificant; however, an exception occurred in the case of patient metabolites being far more abundant than those in control samples. The simplest solution to this problem is to derivatise the patient sample with the non-isotope labelled [.sup.2H.sub.0]-GP reagent (i of Table 1) and the control sample with [.sup.2H.sub.5]-GP (ii of Table 1). However, we have now altered the synthetic method to use [.sup.2H.sub.5]pyridine of 99.96% isotopic purity, essentially eliminating the problem. In fact, the high isotopic impurity of the resultant product with respect to deuterium, in addition to the stability of deuterium on the aromatic ring with respect to H/D exchange are a major advantage of this charge-tag. A similar issue arises with ethyl [1-.sup.13C and 1,2-.sup.13C.sub.2]bromoacetate (both 99% .sup.13C) [.sup.15N]pyridine (98% .sup.15N) and [.sup.2H.sub.5.sup.15N]pyridine used to generate the isobaric mass tags iii, iv, v and vi of Table 1. However, by selecting ions corresponding to fully isotope labelled derivatives for MS/MS or MS.sup.n the low mass isotopomers are excluded from quantitative measurements minimising the problem (FIG. 1).

(31) Charge-tags based on other compounds of formula (I) may be synthesised using a similar method but using an appropriately substituted pyridine as a starting material.

(32) Charge-tags based on Girard T reagent (formula II) may be synthesised in a similar manner but using trimethylamine instead of pyridine.

Example 2—Extraction of Steroids from Plasma and Blood Spots

(33) Plasma (100 μL) was added dropwise to a solution of absolute ethanol (1.05 mL) containing 24(R/S)-[25,26,26,26,27,27,27-.sup.2H.sub.7]hydroxycholesterol (or 24(R/S)-[26,26,26,27,27,27-.sup.2H.sub.6]hydroxycholesterol) and 22(R)-[25,26,26,26,27,27,27-.sup.2H.sub.6]hydroxycholest-4-en-3-one (20 ng of each in 1.05 mL of absolute ethanol) in an ultrasonic bath. After 5 min the solution was diluted to 70% ethanol by addition of 0.35 mL of water, ultrasonicated for a further 5 min and centrifuged at 14 000×g at 4° C. for 30 min. The supernatant was loaded onto a 200 mg Certified Sep-Pak C.sub.18 cartridge (pre-conditioned with 4 mL of absolute ethanol followed by 6 mL 70% ethanol) and allowed to flow at ˜0.25 mL/min. The flow-through was combined with a column wash of 70% ethanol (5.5 mL) to give SPE1-Fr1 containing the oxysterols. A second fraction (SPE1-Fr2) was collected by eluting with a further 4 mL of 70% ethanol before elution of cholesterol and similarly hydrophobic sterols using 2 mL of absolute ethanol (SPE1-Fr3). Each fraction was concentrated under reduced pressure using a vacuum concentrator (ScanLaf, Denmark) (FIG. 8).

(34) Sterols were extracted from blood spots as described in Griffiths et al (Griffiths W J, Wang Y, Karu K, Samuel E, McDonnell S, Hornshaw M, Shackleton C. Clin Chem. 2008 August; 54(8):1317-24)

Example 3—Charge Tagging of Steroids from Plasma or Blood Spots

(35) The steroid-containing fractions from Example 2 were re-constituted in 100 μL propan-2-ol then treated with KH.sub.2PO.sub.4 buffer (1 mL 50 mM, pH 7) containing 3 μL of cholesterol oxidase (2 mg/mL in H.sub.2O, 44 units/mg protein). The reaction mixture was incubated at 37° C. for 1 hr then quenched with 2.0 mL of methanol. Glacial acetic acid (150 μL) was added followed by Girard P reagent (190 mg bromide salt, 150 mg chloride salt, 0.80 mmol). The mixture was vortexed then incubated at room temperature overnight in the dark.

(36) To remove excess reagent from the reaction mixture a recycling method was used. A 200 mg Certified Sep-Pak C.sub.18 cartridge was pre-conditioned with methanol (6 mL), 10% methanol (6 mL) and finally 70% methanol (4 mL). The derivatization mixture from above (3.25 mL in ˜70% organic) was applied to the column and allowed to flow through at ˜0.25 mL/min. The column was washed with 70% methanol (1 mL) followed by 35% methanol (1 mL) and the combined eluent diluted with water (4 mL) to give a solution of ˜9 mL of 35% methanol. This solution was applied to the column, collected, and combined with a column wash of 17.5% methanol (1 mL). Water (9 mL) was added to give a solution in 19 mL of 17.5% methanol which was again applied to the column. The flow-through was collected and the column washed with 10% methanol (2×6 mL). Derivatized steroids were then eluted from the column with methanol (3×1 mL, SPE2-Fr1, Fr2, Fr3) followed by absolute ethanol (1 mL, SPE2-Fr4). Cholesterol was found to be almost exclusively present in SPE2-Fr3 while oxysterols and C.sub.19 steroid sulphates elute in SPE2-Fr1 and Fr2.

Example 4—LC-MS(MSn) on the LTQ-Orbitrap

(37) To analyse GP-tagged sterols, 120 μL from each of SPE2-Fr1 and Fr2 (240 μL in total) were diluted with 160 μL water containing 0.1% formic acid to give a final concentration of 60% methanol. For each experiment, 20 μL was injected onto the LC column and MS, MS.sup.2 and MS.sup.3 spectra recorded as described below.

(38) LC was performed on a Ultimate 3000 HPLC system (Dionex, Surrey, UK) using a Hypersil GOLD revered phase column (1.9 μm particle size, 50×2.1 mm, Thermo Fisher). Mobile phase A consisted of 33.3% methanol, 16.7% acetonitrile and 0.1% formic acid. Mobile phase B consisted of 63.3% methanol, 31.7% acetonitrile and 0.1% formic acid. The chromatographic run started at 20% B for 1 min before increasing the proportion of B to 80% over 7 minutes and maintaining this for a further 5 min. The proportion of B was returned to 20% over 6 s and re-equilibration was for 3 min, 54 s to give a total run time of 17 min. The flow rate was 200 μL/min and the eluent was directed to the atmospheric pressure ionization (API) source of an LTQ-Orbitrap (either an LTQ-Orbitrap XL or Velos). The Orbitrap was calibrated externally before each analytical session and the mass accuracy was better than 5 ppm on the XL and 2 ppm on the Velos. A number of different experimental methods were used.

(39) 1. The first method consisted of a Fourier Transform (FT)-MS scan in the Orbitrap over the m/z range of 400-610 or 300-800 at 30,000 resolution (full width at half-maximum height; FWHM) with a maximum ion fill time of 500 ms. This was followed by MS.sup.2 and MS.sup.3 scans in the linear ion trap (LIT) with maximum ion fill times of 200 ms on the XL or 100 mS on the Velos. Three microscans were performed with the precursor ion isolation width set at 2 on the XL or 1 on the Velos, and the normalised collision energies of 30 for MS.sup.2 and 35 for MS.sup.3 (instrument settings). MS.sup.2 was preferentially performed on [M].sup.+ ions of expected sterols based on a precursor ion inclusion list providing a minimum of 500 ion counts was reached. If a fragment ion corresponding to a neutral loss of the pyridine ring (Py) was observed in the MS.sup.2 event, MS.sup.3 was performed on this ion (providing a minimum of 200 ion counts was reached).

(40) 2. The second experimental method used a multiple reaction monitoring (MRM)-like approach. The Orbitrap® was scanned as described above while selected MS.sup.3 transitions ([M].sup.+.fwdarw.[M-Py].sup.+.fwdarw.) were monitored in the LIT. Two transitions were repetitively monitored over the course of the chromatographic run.

(41) 3. The third experimental utilised the Orbitrap to monitor selected MS.sup.2 transitions ([M].sup.+.fwdarw.[M-Py].sup.+, while the LIT recorded the fragment ions generated in the MS.sup.3 transitions ([M].sup.+.fwdarw.[M-Py].sup.+.fwdarw.). This protocol was only exploited on the LTQ-Orbitrap Velos where the isolation width was set at 1.

Example 5—Validation of Differential and Isobaric Mass Tags

(42) 1. ESI-MS(MS/MS)

(43) For situations where separation of isomers is not a requirement, analysis can be performed by direct-infusion ESI-MS and MS/MS. Validation experiments were initially performed on the Q-TOF mass spectrometer using authentic standards at differing concentrations. Cholesterol and 25-hydroxycholesterol were used to validate the differential mass tag method. Cholesterol tagged with [.sup.2H.sub.0-GP] reagent (i of Table 1) gives an [M].sup.+ ion at m/z 518.4 and when tagged with [.sup.2H.sub.5-GP] (ii of Table 1) the [M].sup.+ ion is shifted to m/z 523.4. Similarly, the [M].sup.+ ion for 25-hydroxycholesterol is at m/z 534.4 when derivatised [.sup.2H.sub.0-GP] (i of Table 1) and at 539.4 when derivatised with [.sup.2H.sub.5]-GP (ii of Table 1). When 1:1 mixtures of sterols derivatised GP (i) and (ii) of Table 1 were analysed by ESI-MS the ratio of peak intensities for ions at 518.4 and 523.4 and for ions of 534.4 and 539.4 were essentially 1:1 (FIG. 2A). With 10:1 and 1:10 mixtures the deviation from the theoretical ratio was less than 20%. The deviation can be explained at least in-part by the inherently limited dynamic range of the Q-TOF Ultima instrument employed.

(44) FIG. 2 illustrates the validation of quantitative charge-tags. FIG. 2(A) shows an ESI-MS recorded on Q-TOF Ultima showing the peak intensity ratio for a 1:1 mixtures of cholesterol (Chol) and 25-hydroxycholesterol (25-HC) labelled with [.sup.2H.sub.0]-GP (i of Table 1) and [.sup.2H.sub.5]-GP (ii of Table 1).

(45) 2. LC-MS(MS).sup.n on the LTQ-Orbitrap

(46) To validate the concept of differential mass tags for steroid analysis by LC-MS, aliquots of control plasma were analysed on the LTQ-Orbitrap at volume ratios 1:1, 2:1, 3:1 5:1, 10:1, 1:2, 1:3, 1:5 and 1:10 after labelling with [.sup.2H.sub.0]-GP (i of Table 1) and [.sup.2H.sub.5]-GP (ii of Table 1) reagents, respectively (FIGS. 2B & C). As is shown in FIG. 2C the measured analyte ratios are in good agreement with the theoretical values. To correct for any sample handling errors prior to LC-MS analysis, the common internal standard [26,26,26,27,27,27-.sup.2H.sub.6]24(R/S)-hydroxycholesterol was used throughout. Similar experiments performed with isobaric mass tags which also gave satisfactory ratios over the same concentration range utilizing the MS.sup.2 [M].sup.+.fwdarw.[M-Py].sup.+ and MS.sup.3 [M].sup.+.fwdarw.[M-Py].sup.+.fwdarw. transitions. A 1:10 ratio is illustrated in FIG. 6).

Example 6—Simultaneous Quantification of 3-Oxo- and 3β-Hydroxy-Steroids

(47) Quantitative charge-tags can be used to differentiate between molecules naturally possessing an oxo group and those oxidized by e.g. cholesterol oxidase, to contain one. This allows the profiling of all oxo and 3β-hydroxy steroids in a single analysis as set out in Table S1. Here we illustrate this with infant plasma. Two identical aliquots of infant plasma were worked up in parallel with or without enzymatic oxidation. The oxidised sample was derivatised with [.sup.2H.sub.5]-GP (ii of Table 1), while the non-oxidised sample was derivatised with [.sup.2H.sub.0]-GP (i of Table 1). The samples were analysed by LC-MS using the LTQ-Orbitrap. By plotting reconstructed ion chromatograms (RICs) for molecules derivatised with [.sup.2H.sub.5]-GP (ii) (following cholesterol oxidase treatment) and [.sup.2H.sub.0]-GP (i) (in the absence of cholesterol oxidase) the quantities of 3-oxo compounds were revealed by the intensity of [.sup.2H.sub.0]-GP (i) labelled analytes and the quantities of 3β-hydroxy compounds by the difference in intensity of [.sup.2H.sub.5]-GP (ii) and [.sup.2H.sub.0]-GP (i) labelled analytes. From FIG. 3A it is clear that there is essentially no endogenous 3-oxocholest-4-enoic acid, but a high level of the cholesterol oxidase substrate 3β-hydroxycholest-5-enoic acid. The situation is different for 7α,25- and 7α,26-dihydroxycholesterols which are accompanied in plasma by their down-stream metabolites 7α,25- and 7α,26-dihydroxycholest-4-en-3-ones, and also for 3β,7α-dihydroxycholest-5-enoic acid and its metabolite 7α-hydroxy-3-oxocholest-4-enoic acid (FIGS. 3B & 3C). Table S1 contains data for all the metabolites detected in infant and pooled adult plasma. Table S1 shows steroids, oxysterols and cholestenoic acids detected by LC-ESI-MS.sup.n in plasma following SPE and charge-tagging with GP-hydrazine. In the absence of authentic standards presumptive identifications based on exact mass, MS.sup.n spectra and retention time are given. Control values are given in parenthesis.

(48) TABLE-US-00003 TABLE S1 Steroids, Oxysterols and Cholestenoic Acids in Human Plasma. After cholesterol oxidase and GP-tagging Originating structure Met ID Mass Formula Sterol Systematic name Sterol Systematic name (common name) RT AS ng/mL 502.237 C.sub.26H.sub.36N.sub.3O.sub.5S.sup.+ 3β-Hydroxyandrost-5-en-17-one 3β-Hydroxyandrost-5-en-17-one 0.8 Y (264.0) 3-sulphate 17-GP 3-sulphate (Dehydroepiandrosterone) 504.2527 C.sub.26H.sub.38N.sub.3O.sub.5S.sup.+ 3-Hydroxyandrostan-17-one 3-sulphate 3-Hydroxyandrostan-17-one 3-sulphate 0.83 N  (36.9) 17-GP 504.2527 C.sub.26H.sub.38N.sub.3O.sub.5S.sup.+ 3-Hydroxyandrostan-17-one 3-sulphate 3-Hydroxyandrostan-17-one 3-sulphate 1.02 N (154.3) 17-GP 518.2319 C.sub.26H.sub.36N.sub.3O.sub.6S.sup.+ 3β,x-Dihydroxyandrost-5-en-17-one 3β,x-Dihydroxyandrost-5-en-17-one N  (5.9) 3-sulphate 17-GP 3-sulphate 600.3279 C.sub.32H.sub.46N.sub.3O.sub.8.sup.+ 3-Hydroxyandrostan-17-one 3-Hydroxyandrostan-17-one 3- 1.05 N  (22.8) 3-glucuronide 17-GP glucuronide 516.3948 C.sub.34H.sub.50N.sub.3O.sup.+ Cholesta-4,24-dien-3-one 3-GP Cholesta-5,24-dien-3β-ol 10.83 Y (Desmosterol) 516.3948 C.sub.34H.sub.50N.sub.3O.sup.+ Cholesta-4,7-dien-3-one 3-GP Cholesta-5,7-dien-3β-ol 12.07 Y (7-Dehydrocholesterol) 518.4105 C.sub.34H.sub.52N.sub.3O.sup.+ Cholest-4-en-3-one 3-GP Cholest-5-en-3β-ol (Cholesterol) 11.7 Y 520.4261 C.sub.34H.sub.54N.sub.3O.sup.+ 5α-Cholestan-3-one 3-GP 5α-Cholestan-3β-ol (Cholestanol) Y 504.3221 C.sub.31H.sub.42N.sub.3O.sub.3.sup.+ 3-Oxochol-4,6-dien-24-oic acid 3-GP 3-Oxochol-4,6-dien-24-oic acid 3.45 N  (1.4)  38.46 504.3221 C.sub.31H.sub.42N.sub.3O.sub.3.sup.+ 3-Oxochol-4,6-dien-24-oic acid 3-GP 3β-hydroxychol-5,7-dien-24-oic acid 3.45 N  (1.0)  16.08 506.3377 C.sub.31H.sub.44N.sub.3O.sub.3.sup.+ 3-Oxochol-4-en-24-oic acid 3-GP 3-Oxochol-4-en-24-oic acid 4.57 Y (ND)   6.56 506.3377 C.sub.31H.sub.44N.sub.3O.sub.3.sup.+ 3-Oxochol-4-en-24-oic acid 3-GP 3β-hydroxychol-5-en-24-oic acid 4.57 Y  (4.5)  355.29 522.3326 C.sub.31H.sub.44N.sub.3O.sub.4.sup.+ 7α-Hydroxy-3-oxochol-4-en-24-oic 7α-Hydroxy-3-oxochol-4-en-24-oic acid 2.18 Y  (3.9) acid 3-GP  25.57 522.3326 C.sub.31H.sub.44N.sub.3O.sub.4.sup.+ 7α-Hydroxy-3-oxochol-4-en-24-oic 3β,7α-Dihydroxychol-5-en-24-oic acid 2.18 Y  (9.2) acid 3-GP   7.64 532.3898 C.sub.34H.sub.50N.sub.3O.sub.2.sup.+ Cholest-4-ene-3,24-dione 3-GP Cholest-4-ene-3,24-dione 7.91 Y  (3.2)   0.82 532.3898 C.sub.34H.sub.50N.sub.3O.sub.2.sup.+ Cholest-4-ene-3,24-dione 3-GP 3β-Hydroxycholest-5-en-24-one 7.91 Y (ND)  45.05 534.4054 C.sub.34H.sub.52N.sub.3O.sub.2.sup.+ 24S-Hydroxycholest-4-en-3-one 3-GP Cholest-5-ene-3β,24S-diol 7.60 Y  (6.5) (24S-Hydroxycholesterol)  172.60 534.4054 C.sub.34H.sub.52N.sub.3O.sub.2.sup.+ 25-Hydroxycholest-4-en-3-one 3-GP Cholest-5-ene-3β,25-diol 7.91 Y (<5)  (25-Hydroxycholesterol)  398.41 534.4054 C.sub.34H.sub.52N.sub.3O.sub.2.sup.+ 26-Hydroxycholest-4-en-3-one 3-GP Cholest-5-ene-3β,26-diol 8.14 Y  (17.7) ((25R),26-Hydroxycholesterol) 1743.20 534.4054 C.sub.34H.sub.52N.sub.3O.sub.2.sup.+ 7β-Hydroxycholest-4-en-3-one 3-GP Cholest-5-ene-3β,7β-diol 9.84 Y (<1)  (7β-Hydroxycholesterol)  21.00 534.4054 C.sub.34H.sub.52N.sub.3O.sub.2.sup.+ 3β-Hydroxycholest-5-en-7-one 7-GP 3β-Hydroxycholest-5-en-7-one 9.93 Y (<1)  (7-Oxocholesterol)  11.35 534.4054 C.sub.34H.sub.52N.sub.3O.sub.2.sup.+ 7α-Hydroxycholest-4-en-3-one 3-GP Cholest-5-ene-3β,7α-diol 10.39 Y (<1)  (7α-Hydroxycholesterol)  10.21 546.3690 C.sub.34H.sub.48N.sub.3O.sub.3.sup.+ 3-Oxocholest-4,x-dien-26-oic acid 3-GP 3-Oxocholest-4,x-dien-26-oic acid 3 7.35 N  (21.8)  15.55 546.3690 C.sub.34H.sub.48N.sub.3O.sub.3.sup.+ 3-Oxocholest-4,x-dien-26-oic acid 3-GP 3β-Hydroxycholest-5,x-dien-26-oic acid 7.35 N (ND)  150.83 548.3847 C.sub.34H.sub.50N.sub.3O.sub.3.sup.+ x-Hydroxycholest-4-en-3,y-dione 3-GP 3β,x-Dihydroxycholest-5-en-y-one 6.87 N  (5.3) (ND) 548.3847 C.sub.34H.sub.50N.sub.3O.sub.3.sup.+ 3-Oxocholest-4-en-26-oic acid 3-GP 3-Oxocholest-4-en-26-oic acid 7.84 Y (ND)  96.40 548.3847 C.sub.34H.sub.50N.sub.3O.sub.3.sup.+ 3-Oxocholest-4-en-26-oic acid 3-GP 3β-Hydroxycholest-5-en-26-oic acid 7.84 Y (118.4) 4217.53 550.4003 C.sub.34H.sub.52N.sub.3O.sub.3.sup.+ x,y-Dihydroxycholest-4-en-3-one 3-GP Cholest-5-ene-3β,x,y-triol 3.69 N (ND)  105.65 550.4003 C.sub.34H.sub.52N.sub.3O.sub.3.sup.+ x,y-Dihydroxycholest-4-en-3-one 3-GP Cholest-5-ene-3β,x,y-triol 3.93 N (ND)  55.79 550.4003 C.sub.34H.sub.52N.sub.3O.sub.3.sup.+ 24,25-Dihydroxycholest-4-en-3-one Cholest-5-ene-3β,24,25-triol 5.13 Y (ND) 3-GP  409.27 550.4003 C.sub.34H.sub.52N.sub.3O.sub.3.sup.+ 7α,25-Dihydroxycholest-4-en-3-one 7α,25-Dihydroxycholest-4-en-3-one 5.14 Y  (3.1) 3-GP ND 550.4003 C.sub.34H.sub.52N.sub.3O.sub.3.sup.+ 7α,26-Dihydroxycholest-4-en-3-one 7α,26-Dihydroxycholest-4-en-3-one 5.66 Y  (9.8) 3-GP 550.4003 C.sub.34H.sub.52N.sub.3O.sub.3.sup.+ 7α,12α-Dihydroxycholest-4-en-3-one 7α,12α-Dihydroxycholest-4-en-3-one 8.95 Y 3-GP 550.4003 C.sub.34H.sub.52N.sub.3O.sub.3.sup.+ 7α,12α-Dihydroxycholest-4-en-3-one Cholest-5-en-3β,7α,12α-triol 8.95 Y 3-GP 564.3796 C.sub.34H.sub.50N.sub.3O.sub.4.sup.+ x-Hydroxy-3-oxocholest-4-en-26-oic 3β,x-Dihydroxycholest-5-en-26-oic 2.32 N  (13.8) acid 3-GP/x,y-dihydroxycholest-4-en- acid//3β,x,y-trihydroxycholest-5-en-z-one   9.93 3,z-dione 3GP 564.3796 C.sub.34H.sub.50N.sub.3O.sub.4.sup.+ x-Hydroxy-3-oxocholest-4-en-26-oic 3β,x-Dihydroxycholest-5-en-26-oic 2.72 N  (3.8) acid 3-GP/x,y-dihydroxycholest-4-en- acid//3β,x,y-trihydroxycholest-5-en-z-one ND 3,z-dione 3GP 564.3796 C.sub.34H.sub.50N.sub.3O.sub.4.sup.+ 7β-Hydroxy-3-oxocholest-4-en-26-oic 3β,7β-Dihydroxycholest-5-en-26-oic acid 3.55 Y  (15.2) acid 3-GP ND 564.3796 C.sub.34H.sub.50N.sub.3O.sub.4.sup.+ x-Hydroxy-3-oxocholest-4-en-26-oic x-Hydroxy-3-oxocholest-4-en-26-oic 4.77 N (ND) acid 3-GP/x,y-dihydroxycholest-4-en- acid/x,y-dihydroxycholest-4-en-3,z-dione   8.29 3,z-dione 564.3796 C.sub.34H.sub.50N.sub.3O.sub.4.sup.+ x-Hydroxy-3-oxocholest-4-en-26-oic 3β,x-Dihydroxycholest-5-en-26-oic 4.77 N (ND) acid 3-GP/x,y-dihydroxycholest-4-en- acid//3β,x,y-trihydroxycholest-5-en-z-one  36.72 3,z-dione 564.3796 C.sub.34H.sub.50N.sub.3O.sub.4.sup.+ x-Hydroxy-3-oxocholest-4-en-26-oic 3β,x-Dihydroxycholest-5-en-26-oic 5.27 N (ND) acid 3-GP/x,y-dihydroxycholest-4-en- acid//3β,x,y-trihydroxycholest-5-en-  21.73 3,z-dione z-one 564.3796 C.sub.34H.sub.50N.sub.3O.sub.4.sup.+ x-Hydroxy-3-oxocholest-4-en-26-oic 3β,x-Dihydroxycholest-5-en-26-oic 4.6 N  (18.9) acid 3-GP/x,y-dihydroxycholest-4-en- acid//3β,x,y-trihydroxycholest-5-en-z-one ND 3,z-dione 564.3796 C.sub.34H.sub.50N.sub.3O.sub.4.sup.+ 7α-Hydroxy-3-oxocholest-4-en-26-oic 7α-Hydroxy-3-oxocholest-4-en-26-oic acid 6.11 Y (149.4) acid 3-GP  11.23 564.3796 C.sub.34H.sub.50N.sub.3O.sub.4.sup.+ 7α-Hydroxy-3-oxocholest-4-en-26-oic 3β,7α-Dihydroxycholest-5-en-26-oic acid 6.11 Y  (53.7) acid 3-GP ND 566.3952 C.sub.34H.sub.52N.sub.3O.sub.4.sup.+ 7α,12α,x-Trihydroxycholest-4-en-3-one 7α,12α,x-Trihydroxycholest-4-en-3-one 3.87 N 3-GP 580.3745 C.sub.34H.sub.50N.sub.3O.sub.5.sup.+ 7α,x-Dihydroxy-3-oxocholest-4-enoic 7α,x-Dihydroxy-3-oxocholest-4-enoic acid 2.16 N  (1.7) acid 3-GP ND 580.3745 C.sub.34H.sub.50N.sub.3O.sub.5.sup.+ 7α,y-Dihydroxy-3-oxocholest-4-enoic 7α,y-Dihydroxy-3-oxocholest-4-enoic acid 3.6 N   (1.19) acid 3-GP 612.3466 C.sub.34H.sub.50N.sub.3O.sub.5S.sup.+ x-Hydroxycholest-4,y-dien-3-one 3-PG x-Hydroxycholest-4,y-dien-3-one sulphate 5.62 N (ND) sulphate  33.14 612.3466 C.sub.34H.sub.50N.sub.3O.sub.5S.sup.+ x-Hydroxycholest-4,y-dien-3-one 3GP Cholest-5,y-diene-3β,x-diol sulphate 5.62 N (ND) sulphate  22.20 614.3622 C.sub.34H.sub.52N.sub.3O.sub.5S.sup.+ x-Hydroxycholest-4-en-3-one 3GP Cholest-5-ene-3β,x-diol sulphate 6.88 N (ND) sulphate 1496.30 614.3622 C.sub.34H.sub.52N.sub.3O.sub.5S.sup.+ x-Hydroxycholest-4-en-3-one 3GP Cholest-5-ene-3β,x-diol sulphate 7.4 N (ND) sulphate  390.04 630.3571 C.sub.34H.sub.52N.sub.3O.sub.6S.sup.+ x,y-Dihydroxycholest-4-en-3-one 3GP Cholest-5-ene-3β,x,y-triol sulphate 3.96 N (ND) sulphate  28.47 710.44 C.sub.40H.sub.60N.sub.3O.sub.8.sup.+ x-Hydroxycholest-4-en-3-one 3GP Cholest-5-ene-3β,x-diol GlcA 6.47 N (ND) GlcA  173.92 RT = Retention time/min; AS = Authentic standard, Y = Yes, N = No; ND = Not detected;

Example 7—Diagnosis of Oxysterol 7α-hydroxylase Deficiency, CTX and SLOS

(49) The method of Example 6 was essentially repeated, but with a patient and control sample both treated with cholesterol oxidase prior to derivatisation to illustrate the use of the methods of the invention to diagnose oxysterol 7α-hydroxylase deficiency, CTX and SLOS.

(50) FIGS. 4A and 4B relate to the diagnosis of oxysterol 7α-hydroxylase deficiency. This deficiency may be diagnosed by the use of charge tags as illustrated in FIG. 4. Thus, FIG. 4(A) shows RIC of 539.4363 and 534.4054 corresponding to [M].sup.+ ions of monohydroxycholesterols (HC) labelled with [.sup.2H.sub.5]-GP (ii) from patient plasma and with [.sup.2H.sub.0]-GP (i) from control plasma. Both chromatograms are plotted on an identical intensity scale. FIG. 4(B) shows RIC of 553.4155 and 548.3847 corresponding to [M].sup.+ ions of 3β-hydroxycholest-5-enoic acid (HCA) labelled with [.sup.2H.sub.5]-GP (ii) from patient plasma and with [.sup.2H.sub.0]-GP (i) from control plasma. Both chromatograms are plotted on an identical intensity scale. The lower panels in (A) and (B) are mass spectra recorded at peak apex of 7.47 min and 7.16 min, respectively. The chromatograms presented in (A) and (B) define the inborn error of metabolism to be oxysterol 7α-hydroxylase deficiency.

(51) CTX, like oxysterol 7α-hydroxylase deficiency, can present in early infancy as cholestatic liver disease and in adult life as spastic paraparesis (Clayton, 2011). CTX is a consequence of mutations in the CYP27A1 gene. It is easily diagnosed using quantitative charge-tags by the absence of peaks corresponding to (25R)26-hydroxycholesterol and 3β-hydroxycholest-5-enoic acid in the appropriate RIC (FIG. 4C-4E). Diagnosis can be confirmed by high levels of 7α-hydroxycholest-4-en-3-one and of 7α,12α-dihydroxycholest-4-en-3-one (FIG. 4C-4F).

(52) SLOS is a genetic defect of cholesterol biosynthesis. The defective enzyme is 7-dehydrocholesterol reductase (Dhcr7) which reduces 7-dehydrocholesterol to cholesterol. In plasma from control populations the level of 7-dehydrocholesterol is usually two-three orders of magnitude lower than that of cholesterol, while in SLOS patients its level is elevated depending on the severity of disease (Griffiths et al., 2008). This is nicely illustrated in the LC-MS RICs for patient and control plasma derivatised with [.sup.2H.sub.5]-GP (ii) and [.sup.2H.sub.0]-GP (i), respectively; and in the comparative total ion chromatograms (TICs) for the MS.sup.3 transitions [M].sup.+.fwdarw.[M-Py].sup.+.fwdarw. for plasma samples from a control and SLOS patient similarly derivatised (FIGS. 5A & 5B). For a disease such as SLOS diagnosis can be achieved from blood spots on filter paper in the absence of LC separation where the enhanced level of 7-dehydrocholesterol is evident from the ES-MS. Using this simplest methodology it is even possible to determine the severity of the disease

Example 8—Comparing Control, MCI and AD Patient Plasma

(53) About a quarter of the body cholesterol is found in brain (Dietschy and Turley, 2004 J. Lipid Res. 45, 1375-1397), hence it is not surprising that cholesterol, its precursors and metabolites have been suggested as markers of AD disease (Griffiths and Wang, 2009, Eur. J. Lipid Sci. Technol. 111, 14-38). As a prelude to analyzing a large batch of plasma samples from controls, AD and MCI patients and determining their individual steroid profiles we have analysed three pooled samples representing these three groups. Steroids found to show differential abundance in the three samples are isomers of androstanolone 3-sulphate (FIG. 7A). Interestingly, their level falls from control to MCI to AD patients (FIG. 7B). This suggests that levels of isomers of androstanolone 3-sulphate may represent potential markers for the progression to MCI and subsequently AD. Current studies are now being performed to confirm this finding with individual plasma samples.