Welcome to LookChem.com Sign In|Join Free

Article Doi

Identification of unusual oxysterols and bile acids with 7-oxo or 3,5,6-trihydroxy functions in human plasma by charge-tagging mass spectrometry with multistage fragmentation

DOI: 10.1194/jlr.D083246

Source and publish data:

Journal of Lipid Research p. 1058 - 1070 (2018)

Update date:2022-08-17

Topics:

Authors:

Griffiths, William J.Griffiths, William J.

Gilmore, IanGilmore, Ian

Yutuc, EylanYutuc, Eylan

Abdel-Khalik, JonasAbdel-Khalik, Jonas

Crick, Peter J.Crick, Peter J.

Hearn, ThomasHearn, Thomas

Dickson, AlisonDickson, Alison

Bigger, Brian W.Bigger, Brian W.

Wu, Teresa Hoi-YeeWu, Teresa Hoi-Yee

Goenka, AnuGoenka, Anu

Ghosh, ArunabhaGhosh, Arunabha

Jones, Simon A.Jones, Simon A.

Wang, YuqinWang, Yuqin

Read Full Text PDF DownLoad Join now for total 90,000,000 free articles

Article abstract of DOI:10.1194/jlr.D083246

7-Oxocholesterol (7-OC), 5,6-epoxycholesterol (5,6-EC), and its hydrolysis product cholestane-3,5,6-triol (3,5,6-triol) are normally minor oxysterols in human samples; however, in disease, their levels may be greatly elevated. This is the case in plasma from patients suffering from some lysosomal storage disorders, e.g., Niemann-Pick disease type C, or the inborn errors of sterol metabolism, e.g., Smith-Lemli-Opitz syndrome and cerebrotendinous xanthomatosis. A complication in the analysis of 7-OC and 5,6-EC is that they can also be formed ex vivo from cholesterol during sample handling in air, causing confusion with molecules formed in vivo. When formed endogenously, 7-OC, 5,6-EC, and 3,5,6-triol can be converted to bile acids. Here, we describe methodology based on chemical derivatization and LC/MS with multistage fragmentation (MSn) to identify the necessary intermediates in the conversion of 7-OC to 3-hydroxy-7-oxochol-5-enoic acid and 5,6-EC and 3,5,6-triol to 3,5,6-trihydroxycholanoic acid. Identification of intermediate metabolites is facilitated by their unusual MSn fragmentation patterns. Semiquantitative measurements are possible, but absolute values await the synthesis of isotope-labeled standards.—Griffiths, W. J., I. Gilmore, E. Yutuc, J. Abdel-Khalik, P. J. Crick, T. Hearn, A. Dickson, B. W. Bigger, T. H-Y. Wu, A. Goenka, A. Ghosh, S. A. Jones, and Y. Wang. Identification of unusual oxysterols and bile acids with 7-oxo or 3,5,6-trihydroxy functions in human plasma by charge-tagging mass spectrometry with multistage fragmentation.

View More

Full text of DOI:10.1194/jlr.D083246

Identification of unusual oxysterols and bile acids with 7-oxo or 3β,5α,6β-trihydroxy functions in human  
plasma by charge-tagging mass spectrometry with multistage fragmentation  
1
1
1
1
1
1
William J. Griffiths *, Ian Gilmore , Eylan Yutuc , Jonas Abdel-Khalik , Peter J. Crick , Thomas Hearn , Alison  
1
2
3
3
3
3
Dickson , Brian W. Bigger , Teresa Hoi-Yee Wu , Anu Goenka , Arunabha Ghosh , Simon A. Jones and Yuqin  
Wang1*  
1Swansea University Medical School, ILS1 Building, Singleton Park, Swansea SA2 8PP, Wales, UK  
2Stem Cell & Neurotherapies, Division of Cell Matrix Biology and Regenerative Medicine, Stopford Building,  
Oxford Road, University of Manchester, Manchester M13 9PT, UK  
3
th  
Manchester Centre for Genomic Medicine, 6 floor, St Mary’s Hospital, Central Manchester Foundation  
Trust, University of Manchester, Oxford Road, Manchester M13 9WL, UK  
Corresponding authors: Drs William J. Griffiths and Yuqin Wang, Swansea University Medical School, ILS1  
Building, Singleton Park, Swansea SA2 8PP, Wales, UK. Email: w.j.griffiths@swansea.ac.uk and  
y.wang@swansea.ac.uk  
Running title: Identification of unusual oxysterols and bile acids  
Abbreviations: ChOx, cholesterol oxidase; CTX, cerebrotendinous xanthomatosis; CYP, cytochrome P450;  
GP, Girard P; LALD, lysosomal acid lipase deficiency; LC-MS, liquid chromatography – mass spectrometry;  
n
MRM, multiple reaction monitoring; MS , mass spectrometry with multistage fragmentation; NP, Niemann-  
Pick; RIC, reconstructed ion-chromatogram; SLOS, Smith-Lemli-Opitz syndrome; SPE, solid phase extraction;  
5
3
βH,7O-CA, 3β-hydroxy-7-oxocholest-5-en-(25R)26-oic acid; 3βH,7O-Δ -BA, 3β-hydroxy-7-oxochol-5-enoic  
acid; 3βH,7,24-diO-CA, 3β-hydroxy-7,24-bisoxocholest-5-en-26-oic acid; 3β,5α,6β-triHBA, 3β,5α,6β-  
trihydroxycholanoic acid; 3β,5α,6β-triHCa, 3β,5α,6β-trihydroxycholestan-(25R)26-oic acid; 3β,5α,6β-triol;  
cholestane-3β,5α,6β-triol; 3β,5α,6β,24-tetraHCa, 3β,5α,6β,24-tetrahydroxycholestan-26-oic acid; 3β,5α,6β-  
triH,24O-Ca,  
3β,5α,6β-trihydroxy-24-oxocholestan-26-oic  
β,5α,6β,(25R)26-tretrol; 3β,7α-diHCA, 3β,7α-dihydroxycholest-5-en-(25R)26-oic acid; 3β,7α-diHCA(25S),  
β,7α-dihydroxycholest-5-en-(25S)26-oic acid; 3β,7α,12α-triHCA, 3β,7α,12α-trihydroxycholest-5-en-  
acid;  
3β,5α,6β,26-tetrol,  
cholestane-  
3
3
(25R)26-oic acid; 3β,7α,24S-triHCA, 3β,7α,24S-trihydroxycholest-5-en-(25R)26-oic acid; 3β,7α,25-triHCA,  
3
β,7α,25-trihydroxycholest-5-en-26-oic acid; 3β,7β-diHCA(25S), 3β,7β-dihydroxycholest-5-en-(25S)26-oic  
acid; 3β,22,25-triH,24O-C, 3β,22,25-trihydroxycholest-5-en-24-one; 3β,24-diH,7O-CA, 3β,24-dihydroxy-7-  
oxocholest-5-en-26-oic acid; 5,6-EC, 3β-hydroxycholestan-5,6-epoxide; 6β-HC, cholest-4-ene-3β,6β-diol; 7-  
DHC, cholesta-5,7-dien-3β-ol; 7-OC, 3β-hydroxycholest-5-en-7-one; 7α-HC, cholest-5-en-3β,7α-diol; 7α-  
HCO, 7α-hydroxycholest-4-en-3-one; 7αH,3O-CA, 7α-hydroxy-3-oxocholest-4-en-(25R)26-oic acid; 7αH,3O-  
Δ4-BA, 7α-hydroxy-3-oxochol-4-enoic acid; 7α,12α-diHCO, 7α,12α-dihydroxycholest-4-en-3-one; 7α,24S-  
diHCO, 7α,24S-dihydroxycholest-4-en-3-one; 7α,24S-diH,3O-CA, 7α,24S-dihydroxy-3-oxocholest-4-en-  
(25R)26-oic acid; 7α,25-diH,3O-CA, 7α,25-dihydroxy-3-oxocholest-4-en-26-oic acid; 7α,25-diHC, cholest-5-  
ene-3β,7α,25-triol; 7α,25-diHCO, 7α,25-dihydroxycholest-4-en-3-one; 7α,26-diHCO, 7α,(25R)26-  
dihydroxycholest-4-en-3-one; 22R-HCO, 22R-hydroxycholest-4-en-3-one; 24R/S-HC, cholest-5-en-3β,24R/S-  
diol; 25H-VitD  
3
, 9,10-secocholesta-5Z,7E,10(19)-triene-3S,25-diol; 25H,7O-C, 3β,25-dihydroxycholest-5-en-  
7
-one; 26H,7O-C, 3β,(25R)26-dihydroxycholest-5-en-7-one.  
2
Abstract  
-Oxocholesterol (7-OC), 5,6-epoxycholesterol (5,6-EC) and its hydrolysis product cholestane-3β,5α,6β-triol  
3β,5α,6β-triol) are normally minor oxysterols in human samples, however, in disease their levels may be  
7
(
greatly elevated. This is the case in plasma from patients suffering from some lysosomal storage disorders  
e.g. Niemann Pick disease type C, or the inborn errors of sterol metabolism e.g. Smith-Lemli-Opitz  
syndrome and cerebrotendinous xanthomatosis. A complication in the analysis of 7-OC and 5,6-EC is that  
they can also be formed ex vivo from cholesterol during sample handling in air causing confusion with  
molecules formed in vivo. When formed endogenously 7-OC, 5,6-EC and 3β,5α,6β-triol can be converted to  
bile acids. Here, we describe methodology based on chemical derivatisation and liquid chromatography –  
n
mass spectrometry with multistage fragmentation (MS ) to identify the necessary intermediates in the  
conversion of 7-OC to 3β-hydroxy-7-oxochol-5-enoic acid and 5,6-EC and 3β,5α,6β-triol to 3β,5α,6β-  
trihydroxycholanoic acid. Identification of intermediate metabolites is facilitated by their unusual MSn  
fragmentation patterns. Semi-quantitative measurements are possible, but absolute values await the  
synthesis of isotope-labelled standards.  
Key Words: Sterols; cholesterol/metabolism; Niemann-Pick type C; oxidised lipids; tandem mass  
spectrometry  
3
Introduction  
Until recently 7-oxocholesterol (7-OC), 5,6-epoxycholesterol (5,6-EC) and its hydrolysis product cholestane-  
3
β,5α,6β-triol (3β,5α,6β-triol) were regarded by many as artefacts generated by sample handling of  
cholesterol rich material in air (1-4). For a list of sterol abbreviations see Supplemental Table S1. That view  
has changed with the discovery that 7-OC can be generated enzymatically from the cholesterol precursor 7-  
dehydrocholesterol (7-DHC) by cytochrome P450 (CYP) 7A1 (5) and is abundant in plasma of patients with  
Smith-Lemli-Opitz Syndrome (SLOS), where levels of 7-DHC are high, and cerebrotendinous xanthomatosis  
(CTX) where CYP7A1 is highly expressed (6, 7). Furthermore, in patients with lysosomal storage disorders  
Niemann-Pick (NP) disease types C and B and lysosomal acid lipase deficiency (LALD), 3β,5α,6β-triol is  
elevated in plasma as is 7-OC, despite apparently normal levels of 7-DHC (8-13). For an up-to-date review  
see reference (14). Importantly, recent reports by Clayton and colleagues in London (15) and Ory and  
colleagues in St Louis (16) have documented the presence of the unusual bile acid 3β,5α,6β-  
trihydroxycholanoic acid (3β,5α,6β-triHBA) in plasma of NPC patients, while Alvelius et al and Maekawa et  
al have reported the presence of the sulphuric acid and glycine conjugates of 3β-hydroxy-7-oxochol-5-enoic  
5
acid (3βH,7O-Δ -BA) in urine and plasma of NPC patients (17, 18). The observation of these unusual bile  
5
acids associated with NPC and of 3βH,7O-Δ -BA with other disorders e.g. SLOS (19-21) strongly suggests  
that their precursors 7-OC and 5,6-EC are formed in vivo and are not (only) ex vivo artefacts generated  
through sample handling in air. Both 7-OC and 5,6-EC are dietary oxysterols (22, 23), while 5,6-EC may also  
be formed by environmental ozone in lung (24), representing alternative sources of these molecules in  
healthy individuals. In fact Lyons et al showed that 7-OC was rapidly metabolised by the liver in rats and  
excreted into the intestine mainly as aqueous soluble metabolites, presumably bile acids (22). Pulfer and  
Murphy showed that 5,6-EC was the major cholesterol-derived product formed in the reaction of ozone  
with lung surfactant and that 3β,5α,6β-triol, and more abundant levels of an unexpected metabolite,  
3
β,5α-dihydroxycholestan-6-one were formed from 5,6-EC by lung epithelial cells (24).  
5
To investigate how 7-OC is metabolised in vivo into 3βH,7O-Δ -BA and 5,6-EC and 3β,5α,6β-triol into  
3
β,5α,6β-triHBA, as reported in the accompanying manuscript (Griffiths W.J. et al), we have optimised a  
charge-tagging methodology to specifically identify 7-oxo containing sterols and sterols with a 3β,5α,6β-  
4
triol function using chemical derivatisation and liquid chromatography - mass spectrometry (LC-MS) with  
n
multistage fragmentation (MS ). The resultant method is described below.  
5
Materials and Methods  
Human Samples  
Plasma was from patients diagnosed with lysosomal storage disorders, their siblings or parents. All  
participants or their parents provided written informed consent in accordance with the Declaration of  
Helsinki and the study was conducted with institutional review board approval (REC08/H1010/63). NIST  
standard reference material (SRM1950, Gaithersburg, MD), a pooled plasma sample representative of the  
US population (25), was used as a reference.  
Materials  
Oxysterols and C27 bile acids were from Avanti Polar Lipids Inc. (Alabaster, Al, USA); C24 bile acids were a  
kind gift from Professor Jan Sjövall, Karolinska Institutet, Stockholm, Sweden; 3β,5α,6β-triHBA was a kind  
gift from Professor Douglas F. Covey, Washington University School of Medicine. See Supplemental Table  
S1 for a list of oxysterols and bile acid with their common and systematic names, abbreviations, LipidMaps  
ID and suppliers. Cholesterol oxidase (ChOx) enzyme from Streptomyces sp was from Sigma Aldrich Ltd  
2
2
2
(
Dorset, UK), [ H  
0
]Girard P ([ H  
0
]GP) reagent was from TCI Europe (Zwijndrecht, Belgium), [ H ]GP was  
5
synthesised as described in (26). Reversed-phase Certified Sep-Pak C18 (200 mg) and Oasis HLB (60 mg) solid  
phase extraction (SPE) columns were from Waters Ltd (Elstree, Herts, UK).  
Sample Preparation  
The sample preparation protocol is described in detail in (27) and only differs here by the addition of  
additional deuterium-labelled standards. In brief, plasma (100 µL) was added to absolute ethanol (1.05 mL)  
2
]7-OC ([2H  
]7-OC),  
containing deuterated internal standards including [25,26,26,26,27,27,27- H  
7
7
2
2
2
2
[
25,26,26,26,27,27,27- H  
7
]5α,6-EC ([ H  
7
]5α,6-EC), [25,26,26,26,27,27,27- H  
7
]3β,5α,6β-triol ([ H  
7
]3β,5α,6β-  
2
2
2
triol), [25,26,26,26,27,27,27- H  
7
]7α-hydroxycholesterol ([ H  
7
]7α-HC), [25,26,26,26,27,27,27- H  
7
]24R/S-  
2
2
2
hydroxycholesterol ([ H  
7
]24R/S-HC), [26,26,26,27,27,27- H  
6
]7α,25-dihydroxycholesterol ([ H  
6
]7α,25-diHC),  
2
2
2
[
25,26,26,26,27,27,27- H  
7
]22R-hydroxycholest-4-en-3-one ([ H  
7
]22R-HCO), [26,26,26,27,27,27- H  
6
]25-  
2
2
hydroxyvitamin D  
3
and [25,26,26,26,27,27,27- H  
7
]cholesterol ([ H  
7
]C). The solution was diluted to 70%  
6
ethanol with 0.35 mL of water, sonicated and centrifuged to remove precipitated matter. To separate bile  
acids and oxysterols from cholesterol and similarly hydrophobic sterols, the sample solution was applied to  
a 200 mg Sep-Pack C18 column, cholesterol was absorbed while oxysterols and bile acids eluted in the flow-  
through and column wash (SPE1-Fr1, 7 mL 70% ethanol). After a further column wash (SPE1-Fr2, 4 mL 70%  
ethanol), cholesterol, and sterols of similar hydrophobicity, were then eluted in a separate fraction with  
absolute ethanol (SPE1-Fr3, 2 mL). The oxysterol/bile acid (SPE1-Fr1) and cholesterol-rich (SPE1-Fr3)  
fractions were then each divided into two equal aliquots (A) and (B) and lyophilised. After re-constitution in  
propan-2-ol (100 µL), cholesterol oxidase (ChOx, 0.26 units) in 50 mM phosphate buffer (1 mL), pH 7, was  
o
added to sub-fractions (A). After 1hr at 37 C the reaction was quenched with methanol (2 mL). Sub-  
fractions (B) were treated in an identical manner but in the absence of cholesterol oxidase. Glacial acetic  
2
acid (150 µL) was added to each sub-fraction followed by [ H  
5
]GP as the bromide salt (190 mg) to sub-  
2
fractions (A) and [ H  
0
]GP as the chloride salt (150 mg) to sub-fractions (B). The derivatisation reactions were  
left to proceed overnight in the dark at room temperature. Excess derivatisation reagent was removed by  
SPE using a re-cycling method. Each sub-fraction (now 3.25 mL, 69% organic) was applied to a 60 mg Oasis  
HLB column and was washed with 70% methanol (1 mL) and 35% methanol (1 mL). The combined effluent  
was diluted to 35% methanol and re-cycled through the column. This was repeated with dilution to 17.5%  
methanol, a further re-cycling and the column was finally washed with 10% methanol (6 mL). At this point  
all oxysterols/bile acids, or more hydrophobic sterols, were absorbed on the column while unreacted GP  
reagent elute to waste. Oxysterols/bile acids were then eluted with 100% methanol (2 mL, SPE2-Fr1+2)  
while more hydrophobic sterols with 3 mL of 100% methanol (SPE2-Fr1+2+3). Just prior to LC-MS(MSn)  
analysis equal volumes of sub-fractions (A) and (B) were combined and diluted to 60% methanol ready for  
injection. This allowed the simultaneous analysis of sub-fractions A and B.  
LC-MS(MSn)  
Analysis was performed on an Orbitrap Elite (Thermo Fisher Scientific, Hemel Hempstead, UK) exploiting  
electrospray ionisation. Chromatographic separation was achieved with a reversed-phase Hypersil Gold  
column (1.9 µm particle size, 50 × 2.1 mm, Thermo Fisher) using an Ultimate 3000 LC system (now Thermo  
Fisher Scientific) with the mobile phase and gradient described in (26, 27). To separate some closely eluting  
7
oxysterols/bile acids the usual 17 min gradient was extended to 37 min. The mobile phase composition was  
initially at 80% A (33.3% methanol, 16.7% acetonitrile, 0.1% formic acid), 20% B (63.3% methanol, 31.7%  
acetonitrile, 0.1% formic acid) for 10 min, changed to 50% A, 50% B over the next 10 min, maintained at  
this composition for 6 min, then changed to 20%A, 80% B over the next 3 min. The mobile phase  
composition was held at 20% A, 80% B for a further 3 min before returning to 80% A, 20% B in 0.1 min and  
reconditioning the column for a further 4.9 min.  
For each injection five scan events were performed: one high resolution scan (120,000 full-width at half  
3
maximum height definition at m/z 400) in the Orbitrap analyser in parallel to four MS scan events in the  
linear ion-trap. Quantification was by isotope dilution or by using isotope-labelled structural analogues.  
8
Results  
7
-Oxo Containing Sterols  
7
-OC is a α,β-unsaturated ketone (5-en-7-one) and unlike oxysterols/bile acids without an oxo group will  
react with GP reagent in the absence of cholesterol oxidase, hence this compound and its metabolites  
2
possessing a 7-oxo group will be found in the (B) sub-fraction derivatised with the [ H  
0
]GP reagent (Figure  
+
2
1
B). Like most GP derivatives, 7-oxo compounds give an intense [M-Py] ion upon MS (MS/MS, Figure 1B),  
3
+
+
however, MS ([M] [M-Py] ) fragmentation patterns for the 7-oxo derivatives are unlike those from  
compounds with GP derivatisation at position C-3 (cf. Figure 2B & C). In contrast to 3-oxo compounds, 7-  
+
+
oxo compounds show a prominent pattern of fragment-ions corresponding to [M-Py-43] , [M-Py-59] , [M-  
+
+
Py-90] and [M-Py-98] (m/z 412.4, 396.3, 365.3 and 357.3). The suggested structures of these ions are  
shown in Figure 3 and Supplemental Figure S1A. As can be seen in Figure 3 the neutral-losses are associated  
with the unsaturated diazacyclohexanone ring and water, this is evident by the invariant nature of the  
neutral-loss upon variation of the sterol side-chain or by the incorporation of deuterium atoms in the side-  
2
chain e.g. as in [25,26,26,26,27,27,27- H  
7
]7-OC (cf. Figure 2B & Supplemental Figure S2A). Definitive  
13  
15  
13  
identification of the neutral-losses was achieved by utilising [ C N] and [ C  
2
] isotope-labelled GP reagents  
Figure 3). Neutral-losses, [M-Py-18] , [M-Py-28] and [M-Py-61] common to 7-oxo-5-ene and 3-oxo-4-ene  
derivatives are illustrated in Figure 3 and Supplemental Figure S3A.  
+
+
+
(
7
-OC is well resolved from its isomer 7α-hydroxycholest-4-en-3-one (7α-HCO) in the 17 min  
2
chromatographic gradient and is readily quantified by isotope dilution against [ H  
7
]7-OC (Figure 2A), but 26-  
hydroxy-7-oxocholesterol (26H,7O-C), its CYP27A1 metabolite (28, 29), is only partially resolved from its  
isomer 7α,26-dihydroxycholest-4-en-3-one (7α,26-diHCO) by the 17 min chromatographic gradient,  
although it is well resolved from 25-hydroxy-7-oxocholesterol (25H,7O-C), 7α,12α-dihydroxycholest-4-en-3-  
one (7α,12α-diHCO), 7α,24S-dihydroxycholest-4-en-3-one (7α,24S-diHCO) and 7α,25-dihydroxycholest-4-  
en-3-one (7α,25-diHCO), four further metabolites identified in human plasma. Extending the  
chromatographic gradient to 37 min provides almost base line separation of 26H,7O-C from 7α,26-diHCO  
while maintaining resolution from the other isomers (Figure 2D). In the absence of an ideal isotope-labelled  
9
2
standard, approximate quantification of 26H,7O-C is performed against the internal standard [ H  
7
]22R-HCO,  
taking into account relative response factors.  
The down-stream CYP27A1 metabolite of 26H,7-OC, 3β-hydroxy-7-oxocholest-5-en-26-oic acid (3βH,7O-CA)  
(29) is not resolved from its isomer 7α-hydroxy-3-oxocholest-4-en-26-oic acid (7αH,3O-CA) in either the 17  
3
min or 37 min chromatographic gradients (Figure 4A). However, 3βH,7O-CA gives a MS fragment-ion at  
+
3
m/z 426.3 ([M-Py-59] ) that is not present in the MS spectrum of co-eluting 7αH,3O-CA (Figure 4C - 4E).  
3
Thus, by generating a reconstructed-ion chromatogram (RIC) for m/z 426.3±0.3 from the MS spectra,  
+
+
3
5
4
βH,7O-CA is revealed (Figure 4B). Therefore, multiple reaction monitoring (MRM), [M] [M-Py] [M-Py-  
+
9] , can be used to resolve 3βH,7O-CA from its co-eluting isomer 7αH,3O-CA. The fragment-ion at m/z  
+
3
21.3 ([M-Py-64] ) is present in the MS spectrum of 7αH,3O-CA (Figure 4E) but not 3βH,7O-CA (Figure 4C),  
+
+
+
hence the MRM [M] [M-Py] [M-Py-64] can identify 7αH,3O-CA. By necessity, semi-quantification of  
βH,7O-CA is performed utilising the MRM transition 564.4485.3426.3 and reference to an external  
3
standard.  
In bile acid biosynthesis side-chain shortening occurs in the peroxisome and proceeds through 24-  
hydroxylation of the C27 acid, dehydrogenation to a 24-carbonyl group then beta-oxidation to a C24 acid  
(30). The appropriate metabolites from 7-OC are 3β,24-dihydroxy-7-oxocholest-5-en-26-oic acid (3β,24-  
5
diH,7O-CA), 3β-hydroxy-7,24-bisoxocholest-5-en-26-oic acid (3βH,7,24-diO-CA) and 3βH,7O-Δ -BA.  
3
Although authentic standards are not available for these metabolites, by analogy to the MS spectra of 7-  
OC, 26H,7-OC and 3βH,7O-CA where standards are available, prominent patterns of distinguishing  
+
+
+
+
fragment-ions corresponding to the neutral-losses [M-Py-43] , [M-Py-59] , [M-Py-90] and [M-Py-98] are  
3
5
predicted to be present in the MS spectra of 3β,24-diH,7O-CA, 3βH,7,24-diO-CA and 3βH,7O-Δ -BA. Similar  
3
+
to the analysis of 3βH,7O-CA, a MS RIC for [M-Py-59] i.e. m/z 442.3±0.3, should reveal 3β,24-diH,7O-CA  
3
(
Figure 4F). A fragment-ion at m/z 442.3 is absent from the MS spectra of 7α,24S-dihydroxy-3-oxocholest-  
4
-en-(25R)26-oic (7α,24S-diH,3O-CA) and 7α,25-dihydroxy-3-oxocholest-4-en-26-oic (7α,25-diH,3O-CA)  
acids, two commercially available isomers of 3β,24-diH,7O-CA (Supplemental Figure S2I & S2J). While for  
3
most plasma samples we have analysed in recent times the MS RIC channel for m/z 442.3±0.3 is empty  
(27), when patient samples containing high levels of 7-OC from diseases such as NPC are analysed a peak is  
10  
+
+
+
evident in the MRM chromatogram m/z 580.4501.3442.3 i.e. [M] [M-Py] [M-Py-59] (Figure 4F)  
3
and the underlying MS spectrum is compatible with that predicted for 3β,24-diH,7O-CA (Figure 4G). In the  
extended chromatographic gradient of 37 min, presumptively identified 3β,24-diH,7O-CA is resolved from  
isomeric dihydroxy-3-oxocholest-4-en-26-oic acids (Supplemental Figure S2K). Semi-quantitative  
measurement of 3β,24-diH,7O-CA are made using the extended chromatographic gradient assuming the  
2
same response factor as for 7α,24-diH,3O-CA and using the internal standard [ H  
7
]22R-HCO.  
To-date in none of the plasma samples we have analysed have we observed any chromatographic peaks  
3
+
compatible with 3βH,7,24-diO-CA. In contrast, as with 3β,24-diH,7O-CA, an MS RIC for [M-Py-59] (m/z  
5
5
3
84.3) reveals 3βH,7O-Δ -BA in plasma samples from patients with elevated 7-OC (Figure 5B). 3βH,7O-Δ -  
4
BA is clearly resolved from its isomer 7α-hydroxy-3-oxochol-4-enoic acid (7αH,3O-Δ -BA) in the 17 min  
5
chromatographic gradient (Figure 5A). Semi-quantitative measurement are made for 3βH,7O-Δ -BA using  
4
the extended chromatographic gradient assuming the same response factor as for 7αH,3O-Δ -BA and using  
2
the internal standard [ H  
7
]22R-HCO.  
While GP-derivatised sterols with a 7-hydroxy-3-oxo-4-ene structure give a characteristic pattern of ring  
fragment ions at m/z 151.1 (*b -12), 177.1 (*b ), 179.1 (*b -28) and 231.1 (*c +2-18) (Figures 2C, 2E, 4E,  
C) (31), sterols with a 3β-hydroxy-7-oxo-5-ene structure give a minor fragment-ion at m/z 157.1 which  
probably consists of the unsaturated diazacyclohexanone ring and remnants of the B-ring (Figures 2B, 4C,  
D, 5D). This ion is only minor and of limited diagnostic value.  
1
2
3
2
5
4
Semi-Quantitative Measurements  
Using the methodology described, other than for 7-OC where an isotope labelled standard is available i.e.  
[2  
H ]7-OC, we can only make approximate or semi-quantitative measurements. However, as all the 7-oxo  
7
compounds, except 3βH,7O-CA, are resolved from their 3-oxo isomers in either the 17 min or 37 min  
2
chromatographic gradients, quantification is possible using the isotope-labelled internal standard [ H  
7
]22R-  
5
HCO. In the absence of authentic standards, 3β,24-diH,7O-CA and 3βH,7O-Δ -BA were quantified assuming  
4
the same response factors as for their structural analogues 7α,24-diH,3O-CA and 7αH,3O-Δ -BA. As 3βH,7O-  
11  
+
+
+
CA was not chromatographically resolved from 7αH,3O-CA, the MRM [M ][M-Py] [M-Py-59] was used  
for quantification of the former isomer.  
3
β,5α,6β-Trihydroxysterols  
Sterols with a 3β-hydroxy group and a planar A/B ring system are substrates for cholesterol oxidase, these  
include cholest-5-en-3β-ols and 5α-cholestan-3β-ols (32). 3β,5α,6β-triol is planar and becomes oxidised at  
C-3 and also dehydrated through elimination of the 5α-hydroxy group. The dehydration reaction does not  
go to completion under our experimental conditions, so after derivatisation the GP derivatised triol is  
+
+
+
observed as both [M] and [M-H  
2
O] ions (Figure 1A) (33), the [M-H  
2
O] ion giving the stronger signal and  
3
3
+
+
more informative MS spectrum (Figure 6B). In fact, the MS ([M-H  
2
O] [M-H O-Py] ) spectrum of the  
2
3
+
+
3
β,5α,6β-triol is identical to the MS ([M] [M-Py] ) spectrum of cholest-4-ene-3β,6β-diol (6β-HC)  
confirming dehydration through loss of the 5α-hydroxy group. An unusually prominent fragment-ion  
3
+
+
observed in the MS ([M-H  
2
O] [M-H  
2
O-Py] ) spectrum of 3β,5α,6β-triol is at m/z 383.3, corresponding  
+
+
to [M-H  
2
O-Py-72] (Figure 6B, see also Figure 7). A second unusual neutral-loss [M-H  
2
O-Py-100] gives a  
fragment-ion at m/z 355.3. Both fragment-ions are elevated by 7 Da in the spectrum of the  
2
+
[
25,26,26,26,27,27,27- H  
7
] analogue as is the [M-H O-Py-90] fragment-ion (see Supplemental Figures S4A,  
2
2
S5C & S5D). The availability of [ H  
7
]3β,5α,6β-triol allows quantification by isotope dilution utilising RICs for  
+
[
M-H  
2
O] ions. Similar to 3β,5α,6β-triol, 3β,5α,6β-triHBA, the end product of 3β,5α,6β-triol metabolism (15,  
+
+
3
1
6), gives [M] and [M-H  
2
O] ions after GP derivatisation, the latter of which is dominant. The MS ([M-  
+
+
+
H
2
O] [M-H  
2
O-Py] ) spectrum of 3β,5α,6β-triHBA shows a prominent [M-H  
2
O-Py-72] fragment-ion at  
+
+
m/z 371.3, [M-H  
2
O-Py-90] at m/z 353.2 and the unusual neutral-loss [M-H  
2
O-Py-100] at m/z 343.3 (Figure  
6
D, see also Supplemental Figures S4B & S5I). The particularly prominent neutral loss fragment-ion [M-H  
2
O-  
+
3
Py-72] is common to MS spectra of both 3β,5α,6β-triol and 3β,5α,6β-triHBA and can potentially be used  
+
+
to identify further metabolites with a 3β,5α,6β-trihydroxy structure via [M-H  
2
O] [M-H  
2
O-Py] [M-H O-  
2
+
Py-72] MRM chromatograms (see below). In the absence of an isotope-labelled standard, approximate  
2
quantification of 3β,5α,6β-triHBA is made against the internal standard [ H  
7
]24R/S-HC, taking into account  
relative response factors.  
12  
+
In plasma samples high in 3β,5α,6β-triol (e.g. NPC), a new peak is evident in the RIC for the [M-H O] ion of  
2
cholestanetetrols (m/z 555.4317 ± 5 ppm, 5.19 min in Figure 6E) which is not seen in control plasma. With  
+
both the 17 min and 37 min chromatographic gradients this peak is only partially resolved from the [M] ion  
of 7α,25-dihydroxycholesterol (7α,25-diHC) which has an identical mass. However, chromatographic  
3
resolution is sufficiently good to generate an MS (m/z 555.4471.4) spectrum from the apex of the new  
+
peak which is entirely compatible with that expected for the [M-H  
2
O] ion of cholestane-3β,5α,6β,26-tetrol  
+
(
3β,5α,6β,26-tetrol), showing a prominent fragment-ion at m/z 399.3 i.e. [M-H  
2
O-Py-72] , a distinct ion at  
+
+
m/z 381.3 i.e. [M-H  
2
O-Py-90] and a minor fragment at m/z 371.3 i.e. [M-H  
2
O-Py-100] (Figure 6G, see also  
3
Figure 7). The MS (m/z 555.4471.4) spectrum of closely eluting 7α,25-diHC does not give a fragment-  
ion at m/z 399.3 or 371.3 (Supplemental Figure S5L), so by generating a RIC for the fragment-ion m/z 399.3  
3
+
from the MS (m/z 555.4471.4) chromatogram, 3β,5α,6β,26-tetrol ([M-H O] ) is resolved from 7α,25-  
2
+
+
diHC ([M] , Figure 6F). Other isomers of 7α,25-diHC ([M] ) i.e. the dihydroxycholesterols (diHC) 7α,12α-  
diHC, 7α,24S-diHC, 7α,26-diHC, 7β,25-diHC and 7β,26-diHC are all chromatographically resolved from  
+
3
β,5α,6β,26-tetrol ([M-H  
2
O] ). It is only possible to make semi-quantitative measurement of 3β,5α,6β,26-  
tetrol in the absence of an authentic standard and its incomplete chromatographic resolution from 7α,25-  
+
diHC. Semi-quantification is made based on the [M-H  
2
O] of 3β,5α,6β,26-tetrol against the internal  
2
standard [ H  
6
]7α,25-diHC.  
CYP27A1 is the enzyme likely to introduce the (25R)26-hydroxy group to the sterol side-chain. This enzyme  
could then oxidise the primary alcohol to a carboxylic acid to give 3β,5α,6β-trihydroxycholestan-(25R)26-oic  
acid (3β,5α,6β-triHCa). As discussed above, peroxisomal side-chain shortening of C27 acids proceeds  
through C-24 hydroxylation, C-24 dehydrogenation and β-oxidation, to generate 3β,5α,6β-triHBA as the  
ultimate product. The relevant pathway intermediates would be 3β,5α,6β,24-tetrahydroxycholestan-26-oic  
acid (3β,5α,6β,24-tetraHCa) and 3β,5α,6β-trihydroxy-24-oxocholestan-26-oic acid (3β,5α,6β-triH,24O-Ca).  
By analogy to 3β,5α,6β-triol, 3β,5α,6β,26-tetrol and 3β,5α,6β-triHBA; 3β,5α,6β-triHCa, 3β,5α,6β,24-  
+
+
tetraHCa and 3β,5α,6β-triH,24O-Ca should give [M] and [M-H  
2
O] products upon cholesterol oxidase  
3
+
+
+
treatment and GP derivatisation and the MS spectra of the [M-H  
2
O] ions ([M-H  
2
O] [M-H  
2
O-Py] ]) are  
+
+
O-Py-100]+.  
predicted to show neutral-loss fragment-ions [M-H  
2
O-Py-72] , [M-H  
2
O-Py-90] and[M-H  
2
13  
+
For 3β,5α,6β-triHCa the [M-H O-Py-72] ion has an m/z of 413.3 (see Supplemental Figure S4B). A RIC for  
2
+
+
+
the MRM transition [M-H  
2
O] [M-Py-H  
2
O] [M-H  
2
O-Py-72] (m/z 569.4485.3413.3) reveals a new  
chromatographic peak in samples where the concentration of the 3β,5α,6β-triol is high (e.g. NPC) which is  
3
+
+
at, or below, the detection limit in normal plasma samples (Figure 8B). The MS ([M-H  
2
O] [M-H O-Py] )  
2
spectrum underlying the new chromatographic peak in plasma samples rich in 3β,5α,6β-triol is compatible  
O-Py-72]+,  
with that predicted for 3β,5α,6β-triHCa, showing the predicted neutral-loss fragment-ions [M-H  
2
+
+
[
M-H  
2
O-Py-90] and [M-H  
2
O-Py-100] and is thus assigned to this acid (Figure 8C, see also supplemental  
+
Figure S4B and S5N). This new chromatographic peak does, however, co-elute with that of the [M] ion of  
3
3
β,7β-dihydroxycholest-5-en-(25S)26-oic acid (3β,7β-diHCA(25S)) and a second oxysterol with a probable  
β,22,25-trihydroxycholest-5-en-24-one structure, but neither compound gives a fragment ion at m/z 413.3  
3
+
+
in their MS ([M] [M-Py] ) spectra (Supplemental Figure 5O), unlike well resolved 3β,7α-  
dihydroxycholest-5-en-(25S)26-oic (3β,7α-diHCA(25S)) and 3β,7α-dihydroxycholest-5-en-(25R)26-oic  
(3β,7α-diHCA(25R)) acids where this ion is more abundant (Supplemental Figure S5P). In fact, the 25R and  
3
2
5S epimers give identical MS spectra, but are chromatographically resolved. In the absence of an  
authentic standard of 3β,5α,6β-triHCa only semi-quantification is possible. This can be made using the RIC  
+
for the [M-H  
2
O] ion in samples where co-eluting compounds 3β,7β-diHCA(25S) and 3β,22,25-triH,24O-C  
are minor (and assuming a similar response factor to 3β,5α,6β-triHBA), then determining an appropriate  
response factor for the MRM chromatogram 569.4485.3413.3 and using this for quantification of other  
samples.  
+
For 3β,5α,6β,24-tetraHCa the [M-H  
2
O-Py-72] fragment ion has an m/z of 429.3 (Figure S4B). In the RIC for  
+
+
+
the MRM transition [M-H  
2
O] [M-Py-H  
2
O] [M-H O-Py-72] (m/z 585.4501.3429.3) from samples  
2
where 3β,5α,6β-triol is abundant new peaks appears in both the 17 min (Figure 8E) and 37 min  
3
chromatograms. The underlying MS spectrum of the new peak at 3.5 min in an NPC plasma sample (Figure  
8
G, see also Supplemental Figure S5Q), shows a minor fragment-ion at m/z 429.3 and a more prominent  
+
ion at m/z 383.3, the former corresponding to [M-H  
2
O-Py-72] and the latter [M-H  
2
O-Py-118], a dehydration  
+
product of [M-H  
2
O-Py-100] (see Supplemental Figure S4B). Both fragment ions are essentially absent from  
3
MS spectra of the closely eluting peak (Figure 8D) annotated as 3β,7α,12α-trihydroxycholest-5-en-(25R)26-  
14  
oic acid (3β,7α,12α-triHCA, Supplemental Figure S5R) and its isomers 3β,7α,24-trihydroxycholest-5-en-  
(25R)26-oic (3β,7α,24S-triHCA) and 3β,7α,25-trihydroxycholest-5-en-26-oic acid (3β,7α,25-triHCA). The  
+
+
+
MRM chromatogram [M-H  
2
O] [M-Py-H  
2
O] [M-H  
2
O-Py-118] provides even clearer definition of  
+
3
β,5α,6β,24-tetraHCa (Figure 8F). The chromatographic peak for the [M-H  
2
O] ion of 3β,5α,6β,24-tetraHCa  
is sufficiently resolved in the 37 min gradient to allow semi-quantification against the internal standard  
[2  
H ]24R/S-HC and by assuming a response factor similar to 3β,5α,6β-triHBA.  
7
Semi-Quantitative Measurements  
2
Other than for 3β,5α,6β-triol itself, for which the [ H ] analogue is available, we can only make approximate  
7
or semi-quantitative measurements of 3β,5α,6β-triol containing sterols. An authentic standard of 3β,5α,6β-  
triHBA is available, thus can be used to give approximate quantification. Other than 3β,5α,6β-triHCa, all of  
the 3β,5α,6β-triol containing sterols are sufficiently chromatographically resolved from similarly derivatised  
sterols to allow semi-quantification against added internal standards.  
15  
Discussion  
When 7-OC or 3β,5α,6β-triol is abundant in a sample, whether formed enzymatically or through radical  
reactions, the analyst should consider the possibility of the presence of down-stream metabolites. An  
absence may indicate that the primary metabolites are formed ex vivo, while a presence will indicate  
formation in vivo or perhaps from the diet or environment. By taking plasma samples from patients with  
the lysosomal storage disorder NPC as an example we illustrate how metabolites of 7-OC and 3β,5α,6β-triol  
5
leading to bile acids 3βH,7O-Δ -BA and 3β,5α,6β-triHBA, respectively, can be identified.  
Considering metabolites with a 7-oxo-5-ene structure, the fragment-ion resulting from an [M-Py-59]+  
3
+
+
neutral-loss (Figure 3) and appearing in MS ([M] [M-Py] ) spectra is characteristic and valuable for  
+
+
+
metabolite identification via appropriate MRM ([M] [M-Py] [M-Py-59] ) RICs (Figure 4B & 4F & 5B).  
+
The [M-Py-59] fragment-ion is likely a radical cation stabilised by delocalisation across a conjugated system  
from C3 – C7 and two nitrogen atoms (Figure 3). With respect to sterols containing a 3β,5α,6β-triol  
structure, treatment with cholesterol oxidase and GP derivatisation leads to dehydration through loss of  
+
3
+
+
the 5α-hydroxy group and formation of an [M-H  
2
O] ion. MS ([M-H  
2
O] [M-H O-Py] ) leads to a  
2
+
characteristic neutral-loss fragment-ion [M-H  
2
O-Py-72] (Figure 7). Again, the appropriate MRM, [M-  
+
+
O-Py-72]+ can lead to the identification of 3β,5α,6β-triol-containing  
H
2
O] [M-H  
2
O-Py] [M-H  
2
+
metabolites (Figure 6F & 8B & 8E). The [M-H  
2
O-Py-72] fragment-ion is likely stabilised through  
delocalisation of positive charge across the conjugated double bonds in the A-ring (Figure 7). Besides giving  
+
+
the [M-H O] ion, both 3β,5α,6β-triol and 3β,5α,6β-triHBA give an [M] ion. However, the absence of A/B-  
2
3
+
+
ring unsaturation leads to MS ([M] [M-Py] ) spectra which are less structurally characteristic making  
identification of intermediate metabolites difficult (Supplemental Figures S5A & S5H).  
Although the primary aim of this work was identification of 7-oxo-5-ene- and 3β,5α,6β-triol containing  
metabolites, approximate or semi-quantitative measurements can also be made. Accurate quantification,  
however, awaits further synthesis of authentic standards and their isotope-labelled analogues.  
16  
Acknowledgements  
We are grateful to Professor Jan Sjövall, Karolinska Institutet, Stockholm, Sweden and Professor Douglas F.  
Covey, Washington University School of Medicine, for kind gifts of sterol standards. This work was  
supported by the UK Biotechnology and Biological Sciences Research Council (BBSRC, grant numbers  
BB/I001735/1 and BB/N015932/1 to WJG, BB/L001942/1 to YW) and the Welsh Government’s A4B project.  
AD was supported by a KESS2 award from the Welsh Government and European Social Fund. JAK was  
supported by a PhD studentship from Imperial College Healthcare Charities. AG was supported by  
fellowships from European Society of Paediatric Infectious Diseases and the Medical Research Council  
(MR/N001427/1). Members of the European Network for Oxysterol Research (ENOR,  
http://oxysterols.com/) are thanked for informative discussions.  
17  
References  
1
1
.
Kudo, K., G. T. Emmons, E. W. Casserly, D. P. Via, L. C. Smith, J. St Pyrek, and G. J. Schroepfer, Jr.  
989. Inhibitors of sterol synthesis. Chromatography of acetate derivatives of oxygenated sterols. J Lipid  
Res 30: 1097-1111.  
Schroepfer, G. J., Jr. 2000. Oxysterols: modulators of cholesterol metabolism and other processes.  
Physiol Rev 80: 361-554.  
2
.
3
4
.
.
Bjorkhem, I. 2013. Five decades with oxysterols. Biochimie 95: 448-454.  
Griffiths, W. J., P. J. Crick, and Y. Wang. 2013. Methods for oxysterol analysis: past, present and  
future. Biochem Pharmacol 86: 3-14.  
Shinkyo, R., L. Xu, K. A. Tallman, Q. Cheng, N. A. Porter, and F. P. Guengerich. 2011. Conversion of 7-  
5
.
dehydrocholesterol to 7-ketocholesterol is catalyzed by human cytochrome P450 7A1 and occurs by direct  
oxidation without an epoxide intermediate. J Biol Chem 286: 33021-33028.  
6
.
Bjorkhem, I., U. Diczfalusy, A. Lovgren-Sandblom, L. Starck, M. Jonsson, K. Tallman, H. Schirmer, L.  
B. Ousager, P. J. Crick, Y. Wang, W. J. Griffiths, and F. P. Guengerich. 2014. On the formation of 7-  
ketocholesterol from 7-dehydrocholesterol in patients with CTX and SLO. J Lipid Res 55: 1165-1172.  
7
.
Griffiths, W. J., J. Abdel-Khalik, P. J. Crick, M. Ogundare, C. H. Shackleton, K. Tuschl, M. K. Kwok, B.  
W. Bigger, A. A. Morris, A. Honda, L. Xu, N. A. Porter, I. Bjorkhem, P. T. Clayton, and Y. Wang. 2017. Sterols  
and oxysterols in plasma from Smith-Lemli-Opitz syndrome patients. J Steroid Biochem Mol Biol 169: 77-87.  
8
.
Porter, F. D., D. E. Scherrer, M. H. Lanier, S. J. Langmade, V. Molugu, S. E. Gale, D. Olzeski, R. Sidhu,  
D. J. Dietzen, R. Fu, C. A. Wassif, N. M. Yanjanin, S. P. Marso, J. House, C. Vite, J. E. Schaffer, and D. S. Ory.  
010. Cholesterol oxidation products are sensitive and specific blood-based biomarkers for Niemann-Pick  
C1 disease. Sci Transl Med 2: 56ra81.  
Jiang, X., R. Sidhu, F. D. Porter, N. M. Yanjanin, A. O. Speak, D. T. te Vruchte, F. M. Platt, H. Fujiwara,  
2
9
.
D. E. Scherrer, J. Zhang, D. J. Dietzen, J. E. Schaffer, and D. S. Ory. 2011. A sensitive and specific LC-MS/MS  
method for rapid diagnosis of Niemann-Pick C1 disease from human plasma. J Lipid Res 52: 1435-1445.  
1
0.  
Klinke, G., M. Rohrbach, R. Giugliani, P. Burda, M. R. Baumgartner, C. Tran, M. Gautschi, D. Mathis,  
and M. Hersberger. 2015. LC-MS/MS based assay and reference intervals in children and adolescents for  
oxysterols elevated in Niemann-Pick diseases. Clin Biochem 48: 596-602.  
1
1.  
Pajares, S., A. Arias, J. Garcia-Villoria, J. Macias-Vidal, E. Ros, J. de las Heras, M. Giros, M. J. Coll, and  
A. Ribes. 2015. Cholestane-3beta,5alpha,6beta-triol: high levels in Niemann-Pick type C, cerebrotendinous  
xanthomatosis, and lysosomal acid lipase deficiency. J Lipid Res 56: 1926-1935.  
1
2.  
Boenzi, S., F. Deodato, R. Taurisano, B. M. Goffredo, C. Rizzo, and C. Dionisi-Vici. 2016. Evaluation of  
plasma cholestane-3beta,5alpha,6beta-triol and 7-ketocholesterol in inherited disorders related to  
cholesterol metabolism. J Lipid Res 57: 361-367.  
1
3.  
Romanello, M., S. Zampieri, N. Bortolotti, L. Deroma, A. Sechi, A. Fiumara, R. Parini, B. Borroni, F.  
Brancati, A. Bruni, C. V. Russo, A. Bordugo, B. Bembi, and A. Dardis. 2016. Comprehensive Evaluation of  
Plasma 7-Ketocholesterol and Cholestan-3beta,5alpha,6beta-Triol in an Italian Cohort of Patients Affected  
by Niemann-Pick Disease due to NPC1 and SMPD1 Mutations. Clin Chim Acta 455: 39-45.  
1
4.  
Vanier, M. T., P. Gissen, P. Bauer, M. J. Coll, A. Burlina, C. J. Hendriksz, P. Latour, C. Goizet, R. W.  
Welford, T. Marquardt, and S. A. Kolb. 2016. Diagnostic tests for Niemann-Pick disease type C (NP-C): A  
critical review. Mol Genet Metab 118: 244-254.  
1
5.  
Mazzacuva, F., P. Mills, K. Mills, S. Camuzeaux, P. Gissen, E. R. Nicoli, C. Wassif, D. Te Vruchte, F. D.  
Porter, M. Maekawa, N. Mano, T. Iida, F. Platt, and P. T. Clayton. 2016. Identification of novel bile acids as  
biomarkers for the early diagnosis of Niemann-Pick C disease. FEBS Lett 590: 1651-1662.  
1
6.  
Jiang, X., R. Sidhu, L. Mydock-McGrane, F. F. Hsu, D. F. Covey, D. E. Scherrer, B. Earley, S. E. Gale, N.  
Y. Farhat, F. D. Porter, D. J. Dietzen, J. J. Orsini, E. Berry-Kravis, X. Zhang, J. Reunert, T. Marquardt, H. Runz,  
R. Giugliani, J. E. Schaffer, and D. S. Ory. 2016. Development of a bile acid-based newborn screen for  
Niemann-Pick disease type C. Sci Transl Med 8: 337ra363.  
1
7
1
7.  
-oxygenated bile acid sulfates in a patient with Niemann-Pick disease, type C. J Lipid Res 42: 1571-1577.  
8. Maekawa, M., Y. Misawa, A. Sotoura, H. Yamaguchi, M. Togawa, K. Ohno, H. Nittono, G. Kakiyama,  
Alvelius, G., O. Hjalmarson, W. J. Griffiths, I. Bjorkhem, and J. Sjovall. 2001. Identification of unusual  
T. Iida, A. F. Hofmann, J. Goto, M. Shimada, and N. Mano. 2013. LC/ESI-MS/MS analysis of urinary 3beta-  
18  
sulfooxy-7beta-N-acetylglucosaminyl-5-cholen-24-oic acid and its amides: new biomarkers for the detection  
of Niemann-Pick type C disease. Steroids 78: 967-972.  
1
9.  
Griffiths, W. J., Abdel-Khalik, J., Crick, P.T., Wang. Y. 2016. Unravelling new pathways of sterol  
metabolism. In LE STUDIUM Conference, Lipids, nanotechnology and cancer., Hôtel de Ville de Tours,  
France.  
2
0.  
Wang, Y., and W. J. Griffiths. 2017. Oxysterol Lipidomics in Mouse and Man. In Keystone  
Symposium, Lipidomics and Bioactive Lipids in Metabolism and Disease, Granlibakken Tahoe, Tahoe City,  
California, USA, .  
2
1.  
from in-born errors and cancer. Curr Opin Clin Nutr Metab Care 21: 90-96.  
2. Lyons, M. A., S. Samman, L. Gatto, and A. J. Brown. 1999. Rapid hepatic metabolism of 7-  
ketocholesterol in vivo: implications for dietary oxysterols. J Lipid Res 40: 1846-1857.  
3. Gorassini, A., G. Verardo, S. C. Fregolent, and R. Bortolomeazzi. 2017. Rapid determination of  
Wang, Y., and W. J. Griffiths. 2018. Unravelling new pathways of sterol metabolism: lessons learned  
2
2
cholesterol oxidation products in milk powder based products by reversed phase SPE and HPLC-APCI-  
MS/MS. Food Chem 230: 604-610.  
2
4.  
of cholesterol present in lung surfactant. J Biol Chem 279: 26331-26338.  
5. Quehenberger, O., A. M. Armando, A. H. Brown, S. B. Milne, D. S. Myers, A. H. Merrill, S.  
Pulfer, M. K., and R. C. Murphy. 2004. Formation of biologically active oxysterols during ozonolysis  
2
Bandyopadhyay, K. N. Jones, S. Kelly, R. L. Shaner, C. M. Sullards, E. Wang, R. C. Murphy, R. M. Barkley, T. J.  
Leiker, C. R. Raetz, Z. Guan, G. M. Laird, D. A. Six, D. W. Russell, J. G. McDonald, S. Subramaniam, E. Fahy,  
and E. A. Dennis. 2010. Lipidomics reveals a remarkable diversity of lipids in human plasma. J Lipid Res 51:  
3
2
299-3305.  
6. Crick, P. J., T. William Bentley, J. Abdel-Khalik, I. Matthews, P. T. Clayton, A. A. Morris, B. W. Bigger,  
C. Zerbinati, L. Tritapepe, L. Iuliano, Y. Wang, and W. J. Griffiths. 2015. Quantitative charge-tags for sterol  
and oxysterol analysis. Clin Chem 61: 400-411.  
2
7.  
Abdel-Khalik, J., E. Yutuc, P. J. Crick, J. A. Gustafsson, M. Warner, G. Roman, K. Talbot, E. Gray, W. J.  
Griffiths, M. R. Turner, and Y. Wang. 2017. Defective cholesterol metabolism in amyotrophic lateral  
sclerosis. J Lipid Res 58: 267-278.  
2
8.  
hydroxylase in HepG2 cells. Lipids 36: 701-711.  
9. Heo, G. Y., I. Bederman, N. Mast, W. L. Liao, I. V. Turko, and I. A. Pikuleva. 2011. Conversion of 7-  
Lyons, M. A., and A. J. Brown. 2001. Metabolism of an oxysterol, 7-ketocholesterol, by sterol 27-  
2
ketocholesterol to oxysterol metabolites by recombinant CYP27A1 and retinal pigment epithelial cells. J  
Lipid Res 52: 1117-1127.  
3
7
3
0.  
2: 137-174.  
1. Griffiths, W. J., T. Hearn, P. J. Crick, J. Abdel-Khalik, A. Dickson, E. Yutuc, and Y. Wang. 2017. Charge-  
Russell, D. W. 2003. The enzymes, regulation, and genetics of bile acid synthesis. Annu Rev Biochem  
tagging liquid chromatography-mass spectrometry methodology targeting oxysterol diastereoisomers.  
Chem Phys Lipids 207: 69-80.  
3
2.  
sources, physical properties and analytical applications. J Steroid Biochem Mol Biol 72: 169-195.  
3. Wang, Y., K. M. Sousa, K. Bodin, S. Theofilopoulos, P. Sacchetti, M. Hornshaw, G. Woffendin, K.  
MacLachlan, J., A. T. Wotherspoon, R. O. Ansell, and C. J. Brooks. 2000. Cholesterol oxidase:  
3
Karu, J. Sjovall, E. Arenas, and W. J. Griffiths. 2009. Targeted lipidomic analysis of oxysterols in the  
embryonic central nervous system. Mol Biosyst 5: 529-541.  
19  
Footnote  
Swansea Innovations Ltd have licensed derivatisation technology described in this paper to Avanti Polar  
Lipids Inc and Cayman Chemical.  
20  
Figures  
1
A
SUB-FRACTION A  
MS2  
MS3  
ChOx  
[2H ]GP  
5
HO  
OH  
O
O
O
OH  
N
OH  
N
OH  
D
C
Chemical Formula: C27H46O2  
Exact Mass: 402.3498  
Chemical Formula: C27H44O2  
Exact Mass: 400.3341  
HN  
HN  
N
CD  
CD  
O
DC  
[M]+  
O
[M-Py]+  
7
-HC  
C
D
Chemical Formula: C29H47N2O2+  
Chemical Formula: C34H47D5N3O2+  
Exact Mass: 539.4368  
Exact Mass: 455.3632  
MS2  
MS3  
ChOx  
[2H ]GP  
5
HO  
N
N
[M-Py]+  
OH  
OH  
OH  
D
C
OH  
[M]+  
OH  
OH  
NH  
OH  
HN  
OH  
DC  
DC  
N
Chemical Formula: C27H46O3  
Exact Mass: 418.3447  
Chemical Formula: C34H49D5N3O3+  
Exact Mass: 557.4474  
Chemical Formula: C29H49N2O3+  
Exact Mass: 473.3738  
Chemical Formula: C27H48O3  
Exact Mass: 420.3603  
CD  
O
O
C
D
3
,5,6-triol  
ChOx  
[2  
MS2  
MS3  
H5]GP  
N
N
[M-H2O-Py]+  
[M-H2O]+  
D
OH  
C
NH  
OH  
HN  
OH  
DC  
DC  
N
Chemical Formula: C27H44O2  
Exact Mass: 400.3341  
Chemical Formula: C34H47D N O  
+
Chemical Formula: C29H47N O  
+
5
3
2
2
2
CD  
O
O
Exact Mass: 539.4368  
Exact Mass: 455.3632  
C
D
1
B
SUB-FRACTION B  
MS2  
MS3  
[2  
H0]GP  
N
OH  
N
OH  
O
OH  
Chemical Formula: C27H44O2  
Exact Mass: 400.3341  
HN  
HN  
[M-Py]+  
N
[M]+  
Chemical Formula: C29H47N2O2+  
O
O
7-HCO  
Chemical Formula: C34H52N3O2+  
Exact Mass: 455.3632  
Exact Mass: 534.4054  
[2  
MS3  
H0]GP  
MS2  
HO  
O
HO  
N
HO  
N
Chemical Formula: C27H44O2  
Exact Mass: 400.3341  
NH  
NH  
N
[M]+  
[M-Py]+  
O
O
7
-OC  
Chemical Formula: C34H52N O  
+
3
2
Chemical Formula: C29H47N2O2+  
Exact Mass: 455.3632  
Exact Mass: 534.4054  
Figure 1. (A) Oxidation of 3β-hydroxysterols with cholesterol oxidase (ChOx) then derivatisation with  
2
n
2
n
[
H
5
]GP and MS fragmentation. (B) Derivatisation of oxosterols with [ H ]GP and MS fragmentation.  
0
21  
7
-OC  
7α,26-diHCO  
2D
2
A
100  
8.91 26H,7O-C  
8.28  
9
.88  
10  
0
RIC: 534.4054  
RIC: 550.4003  
NPC  
7
-OC, 135 ng/mL  
NPC  
7α-HCO  
7α,25-diHCO  
7
α,25-diHCO  
7α,26-diHCO  
26H,7O-C, 2.5 ng/mL  
6.54  
%
RA
%RA
12.89  
1
0.74  
7α,12α-diHCO  
10.24  
9.58  
21.57  
0
0
100
100  
8.07  
RIC: 534.4054  
NIST SRM1950  
10.24  
7α-HCO  
7α,26-diHCO  
RIC: 550.4003  
NIST SRM1950  
7-OC, <5 ng/mL  
26H,7O-C  
7α,25-diHCO  
7α,26-diHCO  
8.63  
7α,25-diHCO  
26H,7O-C, <1 ng/mL  
7
-OC  
%
RA
7
α-HCO  
%RA  
6.24  
9.90  
1
0.68  
7α,12α-diHCO  
12.67  
9.33  
21.50  
0
0
0
1
2
2
3
4
4
5
6
6
7
8
9
10  
11  
12  
12  
13  
14  
14  
15  
16  
0
0
2
4
4
6
8
8
10  
12  
12  
14  
16  
18  
20  
20  
22  
24  
24  
26  
28  
28  
30  
0
8
10  
1
6
min  
min  
M-Py-28  
427  
2
B
2E
3
3
M-Py-18  
MS : 534455  
M
S : 550471  
9.88 min (NPC)  
8.28 min (NPC)  
100  
7-OC  
7α,26-diHCO  
453  
M-Py-43  
100  
412  
M-Py-90  
%
RA
M-Py-18  
%RA
[M]+  
M]+  
[
4
37  
M-Py-28  
M-Py-98 M-Py-59  
M-Py-61  
365 396  
443  
*c  
2
+2-18  
31  
410  
394  
357  
1 3  
*b -12 *b -28  
M-Py-90  
381  
2
425  
428  
1
57  
285  
329  
151  
225 253  
179  
399  
0
1
200  
250  
450  
0
200  
250  
350  
50  
3
0
0
350  
400  
150  
3
0
0
400  
450  
m/z  
m/z  
2
C
M
1
S
3
: 534455  
0.24 min (NPC)  
7α-HCO  
M-Py-18  
37  
M-Py-28  
443  
2
F
3
M
S
:
5
5
0
4
7
1
4
8.91 min (NPC)  
1
0
0
1
0
0
26H,7O-C  
M-Py-18  
453  
%
RA
%RA
[M]  
+
M-Py-28  
[M]  
+
M-Py-43  
M-Py-59  
M-Py-61  
427  
428  
*b -12  
1
394  
M-Py-90  
M-Py-98  
381  
*
b
3
-28  
2
*c +2-18  
M-Py-90  
65  
4
12  
*e’-18  
25  
412  
151  
398  
231  
3
409  
155  
197  
373  
179  
3
252  
301  
0
140  
160  
180  
200  
220  
240  
260  
280  
300  
320  
340  
350  
360  
380  
400  
420  
440  
460  
0
140  
150  
160  
180  
200  
200  
220  
240  
260  
280  
300  
320  
340  
350  
360  
380  
400  
400  
420  
440  
460  
150  
200  
250  
3
0
0
400  
450  
250  
3
0
0
450  
m/z  
m/z  
3
+
+
Figure 2. Chromatographic separation and MS ([M] [M-Py] ) spectra of 7-OC, 7α-HCO, 26H,7O-C and  
2
7
α,26-diHCO from an NPC plasma sample derivatised with [ H ]GP reagent. (A) Reconstructed ion  
0
chromatograms (RICs, m/z 534.4054 ± 5 ppm) demonstrating the separation of 7-OC from 7α-HCO in a  
plasma sample from an NPC (upper panel) patient and in the NIST reference material (lower panel). MS3  
spectra of (B) 7-OC and (C) 7α-HCO from the NPC plasma sample. (D) RIC (550.4003 ± 5 ppm)  
demonstrating chromatographic separation of 26H,7O-C from 7α,26-diHCO and other isomers in the NPC  
(
upper panel) and NIST (lower panel) plasma samples using the 37 min chromatographic gradient. The  
chromatograms in (D) were recorded on different days resulting in an offset in retention time of 0.2 – 0.3  
min in the earlier peaks. MS spectra of (E) 7α,26-diHCO and (F) 26H,7O-C from the NPC sample. Measured  
3
concentrations of 7-OC and 26H,7-OC are given in the right-hand corners of the chromatograms (A) and (D),  
respectively. Note, 7α-HCO, 7α,25-diHCO and 7α,26-diHCO give twin peaks corresponding to syn and anti  
3
2
conformers of the derivative. MS spectra of authentic standards of [ H  
7
]7-OC, 7-OC, 7α-HCO, 26H,7O-C,  
7
α,26-diHCO, 7α,25-diHCO and 7α,24S-diHCO can be found in Supplemental Figure S2A-G.  
22  
3
3
+
+
Figure 3. Patterns of MS ([M] [M-Py] ) neutral-losses which distinguish between, or are common to,  
2
[
H ]GP derivatised 7-oxo-5-ene and 3-oxo-4-ene sterols. Structures of R groups are shown within brackets,  
in the lower left-hand box. Isotope-labelled [ C ]GP and [ C N]GP reagents used to determine the  
0
13  
13 15  
2
composition of the fragment-ions are shown in the lower right-hand box. An asterisk indicates a heavy  
isotope label.  
23  
M-Py-18  
467  
7
αH,3O-CA(25R)/3βH,7O-CA  
4E
4
A
3
MS : 564485  
100  
5.61  
7αH,3O-CA(25R)  
100  
RIC: 564.3796  
NPC  
Authentic standard  
7
αH,3O-CA(25S)  
%
RA  
7
αH,3O-CA(25R)  
5.39  
7αH,3O-CA+  
6
.46  
3βH,7O-CA, 128 ng/mL  
0
100  
5.54  
7αH,3O-CA(25R)  
RIC: 564.3796  
NIST SRM1950  
%RA
M-Py-28  
%
RA  
7
αH,3O-CA(25S) 6.45  
7αH,3O-CA+  
[M]+  
M-Py-61  
5.31  
3βH,7O-CA, 63 ng/mL  
457  
0
424  
421  
100  
5.56  
3βH,7O-CA(25R)  
RIC: 564.3796  
*b -12  
1
449  
439  
*b -28  
3
*c  
+2-18  
M-Py-90  
3βH,7O-CA  
2
%
RA  
151  
Authentic standard  
3
95  
231  
378 406  
179  
0
0
0
1
2
2
3
4
4
5
6
6
7
8
9
10  
11  
12  
12  
13  
14  
14  
15  
16  
140  
160  
180  
200  
220  
240  
260  
280  
300  
320  
340  
360  
380  
400  
420  
440  
460  
480  
500  
0
10  
8
150  
200  
250  
300
350  
400  
450  
500  
min  
m/z  
3
βH,7O-CA(25R)  
3β,24-diH,7O-CA  
4
B
4F
5.62  
MRM: 564485426 
2.35  
1
0
0
10  
0
MRM: 580501
4
4
2
NPC
NPC  
3
βH,7O-CA(25S)  
%
RA  
3βH,7O-CA, 60 ng/mL  
3β,24-diH,7O-CA, 0.5 ng/mL  
5.37  
%RA
0
100  
5.58  
3βH,7O-CA(25S)  
3βH,7O-CA(25R)  
MRM: 564485426 
%
RA  
NIST SRM1950  
0
5.33  
100  
MRM: 580501442  
3
βH,7O-CA, <1 ng/mL  
NIST SRM1950  
0
1
00  
5.57  
3βH,7O-CA(25R)  
RIC: 564485426  
βH,7O-CA  
Authentic standard  
%RA
3
β,24-diH,7O-CA, ND  
%
RA  
3
0
0
0
1
2
2
3
4
5
6
6
7
8
9
10  
11  
12  
12  
13  
14  
14  
15  
16  
1
0
2
2
4
4
6
6
8
10  
12  
12  
14  
16  
0
4
8
10  
0
10  
8
14  
min  
min  
M-Py-18  
467  
4
C
100  
4G
M
S : 580501  
3
M-Py-18  
83  
3
M
3
S
:
5
64485  
2
.
3
5
mi  
n
(NP  
C)  
βH,7O-CA  
4
Authentic standard  
10  
0
3β,24-diH,7O-CA  
%
RA
%RA
M-Py-28  
M-Py-43  
M-Py-59  
M-Py-90  
M-Py-98  
395  
387  
[M]+  
[M]+  
M-Py-28  
M-Py-43  
M-Py-59  
457  
M-Py-90  
426  
442  
439  
442 473  
440  
458  
411  
157  
256  
408  
151  
179  
382  
0
0
1
40  
1
160  
50  
180  
200  
200  
220  
240  
250  
260  
280  
300  
300
320  
340  
360  
380  
400  
420  
440  
460  
480  
500  
500  
200  
400  
500  
350  
400  
450  
150  
250  
300
350  
450  
m/z  
m/z  
M-Py-18  
467  
4
D
100  
M
5
S
3
: 564485  
.62 min (NPC)  
3βH,7O-CA  
%
RA
M-Py-28  
M-Py-43  
+
[M]  
4
57  
M-Py-59  
M-Py-90  
M-Py-98  
442  
426  
3
95  
439  
1
57  
387  
408  
256  
250  
0
1
200  
300
350  
50  
400  
450  
500  
m/z  
+
+
+
Figure 4. MRM chromatograms [M] [M-Py] [M-Py-59] reveal 3βH,7O-CA and 3βH,24-diH,7O-CA in  
NPC plasma. (A) The authentic standard 3βH,7O-CA (lower panel) co-elutes with 7αH,3O-CA (NPC plasma,  
upper panel; NIST plasma, central panel) in the 17 min chromatographic gradient. (B) MRM chromatogram  
(
m/z 564.4485.3426.3) from an NPC plasma sample (upper chromatogram), the NIST control sample  
24  
(central chromatogram) and an authentic standard of 3βH,7O-CA (lower chromatogram). Measured  
concentrations of 7αH,3O-CA + 3βH,7O-CA and of 3βH,7O-CA alone are given on the right-hand side of the  
chromatograms (A) and (B), respectively. 25S and 25R epimers of 7αH,3O-CA each gives twin peaks  
corresponding to syn and anti conformers of the derivative as seen in (A). The twin peaks observed in (B)  
3
from NPC and NIST samples probably correspond to 25S and 25R epimers of 3βH,7O-CA. MS spectra of (C)  
3
βH,7O-CA authentic standard, (D) 3βH,7O-CA from a NPC plasma sample, and (E) 7αH,3O-CA(25R)  
authentic standard. (F) MRM chromatogram (m/z 580.4501.3442.3) from an NPC plasma sample  
upper panel) and the NIST plasma sample (lower panel) generated with the 17 min gradient. Measured  
concentrations of 3β,24-diH,7O-CA are given in the right-hand corners of the chromatograms. ND, not  
(
3
+
+
detected. (G) MS ([M] [M-Py] ) spectrum underlying the major peak at 2.35 min in (F) from NPC  
plasma, identified as of 3β,24-diH,7O-CA. Structures of fragment ions are shown in Supplemental Figure  
S1B. MS spectra of 7αH,3O-CA(25R) from NIST plasma, 7α,24S-diH,3O-CA and 7α,25-diH,3O-CA authentic  
3
standards are shown in Supplemental Figure S2H-J.  
25  
5
3
βH,7O-Δ -BA  
5
A
5
C
3
M-Py-18  
425  
2.18  
RIC: 522.332
MS : 522443  
10  
0
6
1.96 min (NPC)  
NPC  
4
100  
7αH,3O-Δ -BA  
5
3
βH,7O-Δ -BA, 6 ng/mL  
4
1
.96 7αH,3O-Δ -BA  
%
RA
2.60  
M-Py-28  
0
00
1
4
%RA
1.73  
7αH,3O-Δ -BA  
RIC: 522.3326  
415  
+
NIST SRM195
[M]  
0
407  
M-Py-61  
5
3βH,7O-Δ -BA, <0.5 ng/mL  
%
RA
2
.43  
*b -12  
1
*b -28  
382  
397  
3
*c  
2
+2-18  
M-Py-90  
353  
151  
371  
0
0
0
1
2
2
3
4
4
5
6
6
7
8
9
10  
11  
12  
12  
13  
14  
14  
15  
16  
179  
231  
275  
280  
8
10  
0
140  
1
160  
50  
180  
200  
200  
220  
240  
250  
260  
300  
300  
320  
340  
360  
380  
400  
420  
440  
460  
min  
350  
400  
450  
5
3
βH,7O-Δ -BA  
m/z  
5
B
10  
0
2.16  
MRM: 522443
3
8
4
5D  
M
S
3
: 522443  
M-Py-28  
415  
N
P
C
2.16 min (NPC)  
5
5
3
βH,7O-Δ -BA, 6 ng/mL  
10  
0
3βH,7O-Δ  
-BA  
%
RA
0
1
00  
MRM: 522443384  
%RA
NIST SRM1950  
5
[M]+  
M-Py-18  
3βH,7O-Δ -BA, <0.5 ng/mL  
M-Py-43  
M-Py-90  
M-Py-98  
%
RA
425  
400  
M-Py-59  
0
157  
353  
45  
0
2
2
4
4
6
6
8
10  
12  
12  
14  
14  
16  
0
10  
8
3
384  
225 253 273  
min  
0
1
50  
200  
250  
3
0
0
350  
400  
450  
m/z  
+
+
+
5
Figure 5. The MRM chromatogram [M] [M-Py] [M-Py-59] reveals 3βH,7O-Δ -BA in samples rich in 7-  
5
4
OC. (A) RICs for m/z 522.3326 ± 5 ppm corresponding to 3βH,7O-Δ -BA and its isomer 7αH,3O-Δ -BA in NPC  
(
upper panel) and NIST (lower panel) plasma samples. (B) MRM m/z 522.3443.3384.3 chromatograms  
5
from an NPC (upper panel) and NIST (lower panel) plasma sample. Measured concentrations of 3βH,7O-Δ -  
BA are given in the right-hand corners of the chromatograms. The chromatograms were recorded on  
3
+
different days resulting in an offset in retention time of 0.2 min in the early eluting peaks. MS ([M] [M-  
+
Py] ) spectra of the compounds underlying the chromatographic peaks eluting at (C) 1.96 min (7αH,3O-  
4
5
Δ -BA ) in chromatogram (A) and (D) 2.16 min (3βH,7O-Δ -BA) in chromatogram (B).  
26  
7
β-HC10.0710.42  
5,6-EC  
7α-HC  
3β,5α,6β-triol ([M-H O] )  
6
A
6E
7
α,25-diHC 
10.22  
7β-HC  
+
100  
3
β
,5α,6β,26-tetrol 5.88 7α,26-diHC  
9.62 10.53  
10  
0
RIC: 539.4368  
NPC  
2
+
RIC: 555.4317  
([M-H O] )  
2
7α,26-diHC  
NP
C
3β,5α,6β-triol,  
5
3
n
g
/
m
L
2
4S-HC  
5-HC  
4S-HC  
7.63  
7β,26-diHC  
6.83  
3β,5α,6β,26-tetrol, 1.5 ng/m
L
7α-HC  
2
7.50  
7.32  
4.45  
4.10  
%
RA
26-HC  
.87  
%RA
5.19  
7α,12α-diHC  
2
10.65  
7
8.87  
0
0
100
1
00  
RIC: 539.4368  
NIST SRM1950  
24S-HC  
6-HC  
10.25  
7α-HC  
5.86  
7α,26-diHC  
α,26-diHC  
RIC: 555.4317  
2
NIST SRM195
3β,5α,6β,26-tetrol, ND  
0
7.66  
3β,5α,6β-triol, <1 ng/mL  
7.52 7.87  
7
25-HC  
7α-HC  
RA
24S-HC  
7.33  
%
RA
7
α,25-diHC  
%
6.83  
7α,12α-diHC  
1
0.68  
5.27  
8.90  
9.65  
0
0
0
0
1
2
2
3
4
4
5
6
6
7
8
9
10  
11  
12  
12  
13  
14  
14  
15  
16  
2
4
6
12  
14  
0
8
10  
8
10  
min  
min  
M-H  
2
O-Py-28  
6F
6
B
M
1
S
3
: 539455  
0.53 min (NPC)  
3β,5α,6β-triol  
5.21 3β,5α,6β,26-tetrol  
10  
0
MRM: 555471399  
10  
0
427  
([M-H O] )  
+
2
NPC  
5.88 7α,26-diHC  
3β,5α,6β,26-tetrol, 1.5 ng/mL  
%
RA
7α,26-diHC  
6.87  
%
RA
0
100
5.92  
7
α,26-diHC  
MRM: 555471  
3
9
9
O]+  
2
[M-H  
M-H  
2
O-Py-72  
83  
6.85  
NIST SRM1950  
M-H  
2
O-Py-90  
3β,5α,6β,26-tetrol, ND  
3
*b  
2
M-H  
2
O-Py-18  
%RA
M-H O-Py-100  
2
437  
365  
412  
398  
177  
3
55  
229  
325  
0
1
50  
200  
250  
350  
450  
0
2
4
6
12  
14  
3
0
0
400  
0
8
10  
min  
m/z  
5
3
β,7β-diH-Δ -BA  
6
C
6G
M
S
3
: 555471  
M-H  
2
O-Py-28  
443  
1.41  
0
RIC: 527.364
1
0
0
5.2 min (NPC)  
O]+)  
2
10  
0
3β,5α,6β,26-tetrol  
3
β,5α,6α-triHBA ([M-H  
NP
C
3β,5α,6β-triHBA, 3 ng/m
L
1.61  
RA
1.79 3β,7β-diH-Δ -BA  
1
5
%
5
.94 3β,7α-diH-Δ -BA  
5
M-H O-  
Py-18  
2
.63 3β,7α-diH-Δ -BA  
2
%
RA  
0
1
00  
5
1.72  
3β,7α-diH-Δ -BA  
RIC: 527.3640  
[M-H  
2
O]+  
453  
NIST SRM195
0
M-H  
2
O-Py-72  
5
3β,7β-diH-Δ -BA  
3β,5α,6β-triHBA, ND  
2
M-H O-Py-90  
%
RA1.21  
M-H  
2
O-Py-100 399  
5
*b2  
425  
2.42  
3
β,7α-diH-Δ -BA  
371  
381  
177  
0
0
0
0
1
2
3
4
4
5
6
6
7
8
9
10  
11  
12  
12  
13  
14  
14  
15  
16  
140  
150  
160  
180  
200  
220  
240  
250  
260  
280  
300  
320  
340  
360  
380  
400  
420  
440  
460  
2
8
10  
3
0
0
350  
400  
450  
200  
min  
m/z  
2
M-H O-Py-28  
6
D
100  
M
1.  
S : 527 443  
3
6
1
mi  
n
(NP  
C)  
415  
3β,5α,6β-triHBA  
%
RA
[M-H O]  
+
2
2
M-H O-Py-18  
M-H O-Py-72  
2
425  
2
M-H O-Py-90  
*b  
2
3
71  
397  
M-H O-Py-100  
2
4
07  
177  
353  
43  
3
86  
3
313  
0
1
50  
200  
350  
400  
450  
250  
3
0
0
m/z  
+
O-Py-100]+  
Figure 6. 3β,5α,6β-triol, 3β,5α,6β-triHBA and 3β,5α,6β,26-tetrol give [M-H  
2
O-Py-72] and [M-H  
2
3
+
+
neutral-loss fragment-ions in their MS ([M-H  
2
O] [M-H O-Py] ) spectra. (A) RIC, m/z 539.4368 ± 5 ppm,  
O] ions) from hydroxycholesterols  
[M] ions) and 5,6-EC ([M] ions) in NPC (upper panel) and NIST (lower panel) plasma samples. Measured  
2
+
demonstrating chromatographic separation of 3β,5α,6β-triol ([M-H  
2
+
+
(
27  
concentrations of 3β,5α,6β-triol are given in the right-hand corners of the chromatograms.  
Monohydroxycholesterols give syn and anti conformers of the GP derivative, resulting in twin peaks. (B)  
3
+
+
MS ([M-H  
2
O] [M-H  
2
O-Py] ) spectrum of 3β,5α,6β-triol from an NPC plasma sample. (C) RIC of m/z  
+
5
27.3640 ± 5 ppm demonstrating chromatographic separation of 3β,5α,6β-triHBA [M-H  
diH-Δ -BA ([M] ions) and 3β,7α-diH-Δ -BA ([M] ions) in NPC (upper panel) and NIST (lower panel) plasma  
samples. Measured concentrations of 3β,5α,6β-triHBA are given in the right-hand corners of the  
2
O] from 3β,7β-  
5
+
5
+
5
chromatograms. Both diH-Δ -BA isomers give twin chromatographic peaks. The chromatograms were  
3
+
recorded on different days resulting in a retention time shift of ~0.2 min. (D) MS ([M-H  
2
O] [M-H O-  
2
+
Py] ) spectrum of 3β,5α,6β-triHBA in an NPC plasma sample. (E) RIC for m/z 555.4317 corresponding to  
+
+
the [M-H O] ion of cholestanetetrols and the [M] ion of dihydroxycholesterols from NPC (upper panel) and  
2
NIST (lower panel) plasma samples. Measured concentrations of 3β,5α,6β,26-tetrol are given in the right-  
hand corners of the chromatograms. (F) RIC for the MRM transition m/z 555.4471.4399.3  
+
+
+
corresponding to [M-H  
2
O] [M-H  
2
O-Py] [M-H  
2
O-Py-72] for cholestanetetrols from the NPC (upper  
3
panel) and NIST (lower panel) plasma samples. (G) MS spectrum of the peak eluting at 5.2 min in the NPC  
plasma sample. See Supplemental Figure S4A & S4B for assignment of fragment-ions. MS spectra of  
3
2
authentic standards of [ H  
7
]3β,5α,6β-triol, 3β,5α,6β-triol and 5α,6-EC are shown in Supplemental Figure  
S5C – S5E.  
28  
7
3
Figure 7. Patterns of MS neutral-losses which distinguish between, or are common to, 3β,5α,6β-triol-  
containing and 3β,7-dihydroxy-5-ene sterols. The pattern of neutral-losses shown in the red box distinguish  
+
+
between [M-H O] ions of 3β,5α,6β-triols from [M] ions of 3β,7-dihydroxy-5-ene sterols of identical mass.  
2
Neutral-losses/fragment-ion shown in the blue box are common to both structures. Structures of R groups  
are shown within brackets, in the lower left-hand box.  
29  
3
β,7β,24-  
3β,5α,6β,24-tetraHCa([M-H  
O]+)  
3
β,7β-diHCA(25R)  
2
8
A
3.64 3β,7α-diHCA(25S)  
3β,7α-diHCA(25R)  
8D
tr C  
iH
A
RIC: 569.4110  
NPC  
10  
0
2.22  
3.50  
1
00  
RIC: 585.4059  
NPC  
1.48  
3β,7α,12α-triHCA  
3β,5α,6β,24-tetraHCa, 2.5 ng/mL  
3
β,7α-diHCA(25S)  
.56  
3β,7α-diHCA(25R)  
4
.92  
%
RA3β,7β-diHCA  
%RA
5
(
25S)  
4
.68  
6.46  
0
100
0
%RA
1
00  
5.54 3β,7α-diHCA(25R)  
RIC: 569.4110  
NIST SRM1950  
3β,7α,12α-triHCA  
RIC: 585.4059  
NIST SRM1950  
3β,7α-diHCA(25S)  
0
3β,5α,6β,24-tetraHCa, ND  
3β,7α-diHCA(25R)  
100  
2.14  
3
β,7α-diHCA(25S)  
%RA
%
RA  
6.45  
3β,7β-diHCA(25R)  
β,7β-diHCA  
25S)  
4.12  
3
(
5
.31  
4.66  
.45  
3
0
0
2
6
14  
0
1
2
2
3
4
4
5
6
7
8
9
10  
11  
12  
12  
13  
14  
14  
15  
0
6
8
10  
0
4
8
10  
12  
min  
min  
3
β,7α-diHCA(25R)  
8
B
3
β
,
7
α
-d
i
H
C
A
(
2
5
S
)
8E
MRM: 569485413  
3β,5α,6β-triHCa  
MRM: 585501429 
10  
0
+
5
.56  
NPC  
10  
0
2.20  
([M-H  
2
O] )  
3β,5α,6β-triHCa, 16 ng/mL 
NPC 
4.69  
3β,5α,6β,24-tetraHCa, 2.5 ng/mL
3
6
β,7α-diHCA(25S)  
.46 3β,7α-diHCA(25R)  
%
RA
%RA
3β,5α,6β,24-tetraHCa  
+
([M-H  
2
O] )  
3.55  
0
0
1
00  
5.54 3β,7α-diHCA(25R)  
100  
RIC: 585501429 
RIC: 569485413  
NIST SRM1950 
NIST SRM1950  
3β,5α,6β-triHCa, <1 ng/mL  
3β,5α,6β,24-tetraHCa, ND  
3
β,7α-diHCA(25S) 3β,7α-diHCA(25S)  
%
RA
%RA
6.43 3β,7α-diHCA(25R)  
5
.29  
4.63  
0
0
0
1
2
2
3
4
4
5
6
6
7
8
9
10  
11  
12  
12  
13  
14  
14  
15  
16  
0
2
2
4
4
6
6
8
10  
12  
12  
14  
14  
16  
0
10  
8
0
8
10  
min  
min  
8
C
M-H O-Py-28  
8F
3
.
5
5
3β,5α,6β,  
2
4
-
t
e
traHCa  
M
4
S
3
: 569485  
2
+
MRM: 585501383  
([M-H  
2
O] )  
.69 min (NPC)  
2.20  
100  
NPC 
457  
1
0
0
3β,5α,6β-triHCa  
3β,5α,6β,24-tetraHCa, 2.5 ng/mL
M-H O-  
2
%RA
Py-18  
467  
%
RA
0
100  
O]+  
M-H  
2
O-Py-72  
RIC: 585501383 
[M-H  
2
NIST SRM1950 
M-H  
2
O-Py-90  
M-H  
2
O-Py-100  
3β,5α,6β,24-tetraHCa, ND  
385  
%RA  
439  
4
13  
449  
151  
179  
395 428  
0
0
200  
250  
300
350  
400  
500  
0
0
2
2
4
4
6
6
8
10  
12  
12  
14  
14  
16  
150  
450  
8
10  
min  
m/z  
M-H  
2
O-Py-18  
483  
8
G
M
3
S
3
: 585501  
.52 min (NPC)  
3β,5α,6β,24-triHCa  
1
00  
M-H  
2
O-Py-28  
73  
4
%
RA
455  
O-Py-72 465  
2
M-H  
[M-H  
2
O]+  
M-H  
2
O-Py-90  
M-H O-Py-108  
2
M-H  
2
O-Py-118  
437  
411  
4
429  
22  
383  
365  
185  
239  
393  
0
1
50  
200  
250  
300
350  
400  
450  
500  
m/z  
Figure 8. Identification of 3β,5α,6β-triHCa and 3β,5α,6β,24-tetraHCa in plasma samples rich in 3β,5α,6β-  
+
+
triol. (A) RIC of m/z 569.4110 ± 5 ppm corresponding to [M-H  
2
O] and [M] ions of 3β,5α,6β-triHCa and  
dihydroxycholestenoic acids, respectively, from plasma from a patient with NPC (upper panel) and the NIST  
30  

Products guided by the article

Product name:4-CHOLESTEN-7-BETA-OL-3-ONE

Cas No:25876-54-4

25876-54-4

R&D Labs maybe for 25876-54-4

Relevant to this article

Hot Product