June 2023

Medical Mass Spectrometry Vol. 7 No. 1

Short Communication

Simultaneous determination of cholesterol precursors, plant sterols,
and oxysterols in plasma using one-round pretreatment
1

1

2

3, 4

Naoko Kuwabara , Fumiko Fuwa , Shinji Sato , Satoshi Hirayama ,3

1

Takashi Miida , Saori Nakagawa *Laboratory of Bio-analytical Chemistry, Department of Pharmaceutical Sciences,
Niigata University of Pharmacy and Applied Life Sciences, 265‒1 Higashijima, Akiha-ku, Niigata 956‒8603, Japan
2
Laboratory of Functional and Analytical Food Sciences, Department of Applied Life Sciences,
Niigata University of Pharmacy and Applied Life Sciences, 265‒1 Higashijima, Akiha-ku, Niigata 956‒8603, Japan3
Department of Clinical Laboratory Medicine, Juntendo University School of Medicine,2‒1‒1 Hongo, Bunkyo-ku, Tokyo 113‒8421, Japan
4
Center of Health Service, Tokyo Gakugei University, 4‒1‒1 Nukuikita-machi, Koganei-shi, Tokyo 184‒8501, Japan
1

Abstract

Cholesterol is biosynthesized mainly in the liver, absorbed from diets in the small intestine, and metabolized tooxysterols or cholestanol. The measurement of cholesterol, its precursors, plant sterols, cholestanol, and oxysterols in plasma is useful in the diagnosis of inherited disorders of cholesterol biosynthesis, malformation syndromes, familial hypercholesterolemia, cerebrotendinous xanthomatosis or sitosterolemia, and the evaluation of drug administration. Herein, we report the development of a new method for the determination of cholesterol, its related sterols, and oxysterols in plasma by a
one-round pretreatment using gas chromatography coupled with mass spectrometry. Plasma pretreatments were performed
as follows: lipid extraction, sterol ester saponiﬁcation, extraction with n-hexane, and trimethylsilyl (TMS) derivatization. In
the case of oxysterol, pretreatment requires additional solid-phase extraction before TMS to remove a large amount of cholesterol. Cholesterol, ﬁve cholesterol precursors, three plant sterols, cholestanol, and twelve oxysterols were simultaneously
pretreated using this method. The recoveries of the plasma samples were 88.8 to 103.4%. The mean relative standard deviation values of the intra- and inter-day precision were less than 14.2%. This new development method appeared to be congruent with the conventional results, and its accuracy ranged from 85.0 to 115.5%. The concentrations of cholesterol andrelated sterols determined in our study were within the same range as those reported in the previous reports. The developed
pretreatment procedure is simple, useful for application to human plasma, and contributes to the diagnosis of inherited disorders of cholesterol biosynthesis, cholesterol absorption, and other related diseases, and evaluation of drug administration.
Key words: cholesterol, oxysterols, plant sterols, cholesterol precursors, simultaneous determination

Introduction*Corresponding author

Cholesterol is an essential structural component of all

Saori Nakagawa

mammalian cells and is obtained from endogenous andLaboratory of Bio-analytical Chemistry, Department of Phar-

1)exogenous sources . Endogenous cholesterol is synthesized
mainly in the liver from acetyl-coenzyme A and is con-maceutical Sciences, Niigata University of Pharmacy andApplied Life Sciences, 265‒1 Higashijima, Akiha-ku, Niigata

1, 2). There are two path-956‒8603, Japan
Tel/Fax: ＋81‒250‒25‒5296

verted to squalene and lanosterol

Received: January 6, 2023. Accepted: March 2, 2023.
Epub April 7, 2023.

terol and some plant sterols (campesterol, stigmasterol, andβ-sitosterol) are absorbed from the food in the small intes-

DOI: 10.24508/mms.2023.06.005

tine

ways from lanosterol to cholesterol: via lathosterol and2)
7-dehydrocholesterol and via desmosterol (Fig. 1). Choles-

E-mail: saorin@nupals.ac.jp

1)89(Fig. 1). Cholesterol is oxygenized by cytochrome

June 2023

Fig. 1.

Medical Mass Spectrometry Vol. 7 No. 1

Schematic of cholesterol biosynthesis, absorption from the intestine, and metabolic pathways (HMG-CoA; 3-hydroxy-3-methylglutaryl-coenzyme A, HMGCR; HMG-CoA reductase, NPC1 L1; Niemann-Pick C1-like protein 1,
ABCG5; ATP-binding cassette transporter G5, ABCG8; ATP-binding cassette transporter G8, ACAT; acyl-coenzyme
A:cholesterol acyltransferase, TG; triglyceride, MTP; microsomal triglyceride transfer protein, LACS; long-chain
acyl-coenzyme A synthetase, apo B-48; apolipoprotein B-48, ROS; reactive oxygen species, CYP46A1; Cholesterol
24S-hydroxylase, CH25H; Cholesterol 25-hydroxylase, CYP7A1; Cholesterol 7-Alpha-hydroxylase, CYP27A1; Cholesterol 27-hydroxylase, 11 β -HSD1; 11 β -hydroxysteroid dehydrogenase type 1).

P450 (CYP), radical reactions, etc., and produces oxys1)
terols ﬁrst (Fig. 1). Oxysterols are related to several dis-8)

which is associated with cardiovascular disease . However,
ATX is also a common symptom other conditions, such as
9)Achilles tendon thickening, sitosterolemia, and CTX . Sit-eases; for example, 27-hydroxycholesterol, 7-ketocholesterol, 24S-hydroxycholesterol and 7β-hydroxycholesterolosterolemia is an inherited metabolic disorder characterized

could be used as markers of cerebrotendinous xanthomato3)

4)

sis (CTX) , diabetes mellitus , Alzheimerʼs disease

5)

1)

by increased levels of plant sterols such as sitosterol .

andPatients with sitosterolemia require dietary measures

6)lung cancer , respectively.

(restriction of phytosterols in food) and treatment with bile

Several human malformation syndromes, such as lathosterolosis, Smith-Lemli-Opitz syndrome (SLOS), and des-acid sequestrates (colestipol, cholestyramine) or ezetimibe,
9)which are cholesterol absorption inhibitors . CTX has beenreported to not only decreases plasma levels of 27-hydroxy-

mosterolosis, have been associated with defects in cholesterol biosynthesis . Xanthoma of the Achilles tendon(ATX) is a sign of long-term exposure to high blood choles-

cholesterol but to also increases plasma levels of cholesta-

terol in patients with familial hypercholesterolemia (FH),with CTX was used for chenodeoxycholic acid and statins,

7)

3)

nol, lathosterol, campesterol, and sitosterol . Treatment

90

June 2023

Medical Mass Spectrometry Vol. 7 No. 1

plasma by a one-round pretreatment using GC-MS.

which are 3-hydroxy-3-methylglutaryl-CoA reductase inhib9)
itors . Patients with FH frequently have hypercholesterol-

Materials and Methods

emia, whereas patients with CTX and sitosterolemia show
3, 9)

normocholesterolemia

Reagents. FH, sitosterolemia, and CTX

have similar symptoms as ATX; however, they diﬀer in thetreatment and behavior of cholesterol precursors, plant ste-

Methanol, ethyl acetate, n-hexane, potassium hydroxide,
phosphoric acid, squalene and 5α-cholestane {an internalrols, and oxysterols. Therefore, it is important to distin-

standard (IS) for cholesterol, its precursors, plant sterols,

guish these diseases.

and cholestanol)} were purchased from FUJIFILM Wako

Furthermore, statins have been reported to decrease plasma

Pure Chemical Corporation (Osaka, Japan). Lanosterol was

10)oxysterols , decrease the level of lathosterol, and increase
11)

the levels of campesterol and sitosterol compensatory

manufactured by the Nagara Science Corporation (Gifu,
Japan). Lathosterol, 7-dehydrocholesterol, desmosterol,inreported that ezetimibe decreased the levels of campesterol,cholesterol, 7α-hydroxycholesterol, 7β-hydroxycholesterol,4β-hydroxycholesterol, 22S-hydroxycholesterol, choles-

sitosterol, and stigmasterol, increased levels of compensatory
lathosterol, and decreased serum 7β-hydroxycholesterol interol-5β, 6β-epoxide, 22R-hydroxycholesterol, cholesterol5α, 6α-epoxide, 20S-hydroxycholesterol, and 25-hydroxy-Japanese hypercholesterolaemia . Therefore, the simulta-

cholesterol were purchased from Sigma-Aldrich Corporation (St. Louis, USA). 7-Ketocholesterol and 27-hydroxy-

hypercholesterolemic patients. In addition, we previously

12)

neous determination of cholesterol, cholesterol precursors,
plant sterols, cholestanol, and oxysterols in plasma plays ancholesterol were purchased from Avanti Polar Lipids
Incorporated (Birmingham, England). 24S-hydroxycholes-

important role not only in the diagnosis of inherited disorders
1, 7)

of cholesterol biosynthesis
10‒12)

drug administration

terol and 27-hydroxycholesterol-26, 26, 26, 27, 27-D5(27d5-OHC) (IS for oxysterols) were purchased from Medi-but also in the evaluation of.

Several methods for sterol analysis have been pro3, 10, 12‒17). However, to measure cholesterol, its precurposed

cal Isotopes Incorporated (Pelham, USA). Campesterol andβ-sitosterol were purchased from Tama Biochemical Corpo-

sors, plant sterols, cholestanol, and oxysterols, it is neces-

ration Ltd. (Tokyo, Japan) and stigmasterol and β-cholestanol were purchased from Tokyo Chemical Industry Corpo-

sary to use diﬀerent pretreatment methods and machines.
For instance, Mignarri et al.

3)

used a commercial assay kit

ration,(colorimetric) to measure total plasma cholesterol levels,

Ltd.

(Tokyo,Japan).

Tri-Sil

HTP

reagent(hexamethyldisilazane [HMDS] : trimethylchlorosilane[TMCS] : pyridine＝2 : 1 : 10) and a trimethylsilyl derivatiz-high performance liquid chromatography (HPLC) and gas
chromatography coupled to mass spectrometry (GC-MS) to

ing agent, was purchased from Thermo Fisher Scientiﬁcmeasure plasma levels of cholestanol, and GC-MS to mea-

Corporation (Waltham, USA). A Bond Elut SI cartridge

sure plasma levels of cholesterol precursors, plant sterols,(100 mg, 1 mL) was purchased from Agilent Technologies

and oxysterols. This method required three pretreatments

Japan (Tokyo, Japan), Isolute Biotage SI cartridge (100 mg,

18)

developed an

10 mL) was purchased from Biotage Japan (Tokyo, Japan),analytical method for the simultaneous determination ofand human plasma (sample A: Lot. No. R201529M; sample

four cholesterol precursors, three plant sterols, and fouroxysterols in human plasma. This method could not distin-

B: Lot. No. R201537M; sample C: Lot. No. R201557M;
sample D: Lot. No. N113881F, sample E: N113882F, sam-guish between CTX, sitosterolemia, and FH because cho-ple F: N113886F) were purchased from Tennessee Bloodlesterol could not be measured simultaneously. Oxysterols

Service (Memphis, USA).

are present in very low concentrations in all mammals and36
are almost invariably accompanied by a 10 - to 10 -fold

Sample preparation for the conventional method

and three types of machines. Matysik et al.

. Therefore, the simultaneous

The method presented here was compared with a conven-determination of oxysterols requires the removal of a large

tional method of measuring cholesterol precursors, plantamount of cholesterol at pretreatment before trimethylsilyl

sterols, and oxysterols concentrations, as reported by
12)
Hirayama et al. Brieﬂy, to measure markers of choles-

1, 13)

excess of cholesterol(TMS) derivatization.

terol precursors and plant sterols, 50 μL of plasma containing 250 ng of 5α-cholestane (IS) was saponiﬁed for 1 h withThis study aimed to determine the levels of cholesterol,
its precursors, plant sterols, cholestanol, and oxysterols in91

June 2023

Medical Mass Spectrometry Vol. 7 No. 1

2 mL of 10 mol/L potassium hydroxide and 5 mL of metha-

Sample preparation for the newly developed method
The new sample preparation method was based on thenol. After adjusting the pH to 7.0 with 50% phosphoricacid, 5 mL of H2O was added to the mixture and extracted
twice with 10 mL of n-hexane. The n-hexane layer was

method described by Hirayama et al.

12)with modiﬁcations.

Human plasma (250 μL) (samples A‒F) containing 250 ng
of 5α-cholestane and 150 ng of 27d5-OHC (IS) was saponi-

dried under a stream of nitrogen and then derivatized for
30 min at 60°
C with the Tri-Sil HTP reagent. The derivat-

ﬁed for 22 h with 2 mL of 10 mol/L potassium hydroxideized cholesterol precursors and plant sterols were resuspended in 100 μL of n-hexane for GC-MS analysis.

and 10 mL of methanol. After adjusting the pH to 7.0 with
50% phosphoric acid, 5 mL of H2O was added to the mix-

For oxysterols, 250 μL of plasma containing 150 ng
27d5-OHC (IS) was saponiﬁed for 15 min and extracted

ture and extracted twice with 10 mL of n-hexane. For cho-with n-hexane. The n-hexane layer was loaded onto a Bondlesterol, its precursors, plant sterols, and cholestanol, 2 mLof the n-hexane layer was dried under a stream of nitrogen

Elut SI cartridge (100 mg, 1 mL), which was preconditioned
with n-hexane. Excess cholesterol was removed by washingand then derivatized for 30 min at 60°
C with Tri-Sil HTP
reagent. These were resuspended in 100 μL n-hexane forwith n-hexane (2 mL) and ethyl acetate (10%) with n-hex-

GC-MS analysis (Fig. 2). For oxysterols, 18 mL of then-hexane layer was loaded onto an Isolute Biotage SI car-ane. The oxysterols retained on the cartridge were eluted
using ethyl acetate (2 mL). The ethyl acetate eluate wastridge (100 mg, 10 mL) that was preconditioned using
n-hexane. Excess cholesterol was removed by washing withdried under nitrogen and derivatized for 30 min at 60°
Cusing Tri-Sil HTP reagent. The derivatized oxysterols were
resuspended in 50 μL n-hexane for GC-MS analysis.

Fig. 2.

n-hexane (2 mL) and ethyl acetate (10%) with n-hexane.
The oxysterols retained on the cartridge were eluted using

Pretreatment procedure for human plasma for GC-MS analysis (IS, internal standard; 27d5-OHC, 27-hydroxycholesterol-26,26,26,27,27-D5; MeOH, methanol; KOH, potassium hydroxide; RT, room temperature; H3PO4, phosphoricacid; AcOEt, ethyl acetate; GC-MS, gas chromatography-mass spectrometry).
92

June 2023

Medical Mass Spectrometry Vol. 7 No. 1

Table 1. Selected ion monitoring (SIM) of oxysterol derivatives by GC-MS
Sterols

perature, 230°
C; ion source temperature, 250°
C; interface
temperature, 250°
C; injection volume, 1 μL (for cholesterol,m/z

its precursors, plant sterols, and cholestanol) or 5 μL (foroxysterols); detection mode, selected ion monitoring (SIM);
GC-MS instrument, GCMS-QP2010Plus (Shimadzu Corpo-

Quantities ion

Certiﬁcate ion

Cholesterol

368

458, 329

Cholestanol

445

355, 460

Squalene

69

81

Lanosterol

393

498, 483

Lathosterol
7-dehydrocholesterol

458

459, 255

325

351

Desmosterol

343

456, 253

217 for 5α-cholestane (IS), m/z 456 for 7α-hydroxycholesterol, m/z 456 for 7β-hydroxycholesterol, m/z 456 for 4βhydroxycholesterol, m/z 173 for 22S-hydroxycholesterol, m/z 474 for cholesterol-5β, 6β-epoxide, m/z 173 for 22R-hy-

ration, Kyoto, Japan); ion quantities: m/z 69 for squalene,
m/z 393 for lanosterol, m/z 458 for lathosterol, m/z 325 for
7-dehydrocholesterol, m/z 343 for desmosterol, m/z 368 for

Cholesterol precursors

cholesterol, m/z 445 for cholestanol, m/z 382 for campesterol, m/z 83 for stigmasterol, m/z 396 for β-sitosterol, m/z

Plant sterols
Campesterol

382

343

Stigmasterol
β-sitosterol

83

484, 485

396

357

droxycholesterol, m/z 474 for cholesterol-5α, 6α-epoxide,m/z 201 for 20S-hydroxycholesterol, m/z 413 for 24S-hydroxycholesterol, m/z 131 for 25-hydroxycholesterol, m/z
472 for 7-ketocholesterol, m/z 456 for 27-hydroxycholes-

Oxysterols
7α-hydroxycholesterol

IS

456

233

7β-hydroxycholesterol
4β-hydroxycholesterol

456

233

456

356

Cholesterol-5β,6β-epoxide
Cholesterol-5α,6α-epoxide

457385, 459

474

366, 384

24S-hydroxycholesterol
25-hydroxycholesterol

413

145

131

271, 327, 456

7-ketocholesterol
27-hydroxycholesterol

472

367, 382

456

417

terol, and m/z 461 for 27d5-OHC (IS) (Table 1).

Results and Discussion

Chromatograms of cholesterol, its precursors, plant sterols, cholestanol, and oxysterols are shown in Supplementary Fig. S1. The peaks corresponding to ten analytes (cholesterol, its precursors, plant sterols, and cholestenol) and 5α-cholestane (IS) were clearly separated at a concentration

22S-hydroxycholesterol
22R-hydroxycholesterol

173

546, 531

173

546, 531

of 5 μg/mL (Fig. S1A) except for 7-dehydrocholesterol,

20S-hydroxycholesterol

201

461

whose peak shape diﬀered from that of the other sterols.
The smaller ion intensity of 7-dehydrocholesterol, as indi-

5α-cholestane27d5-OHC

217

357

461

422

cated by its mass spectrum, presumably led to its higher
limit of quantitation than that of the other sterols. Therefore, the chromatogram of 7-dehydrocholesterol corresponded to a concentration of 10 μg/mL (Fig. S1A). Simi-

GC-MS, gas chromatography-mass spectrometry; IS, internal
standard; 27d5-OHC, 27-hydroxycholesterol-26,26,26,27,27-D5

larly,

thepeaks

ethyl acetate (2 mL). After drying the elute under a stream

corresponding to twelve analytes(oxysterols) and 27d5-OHC (IS) were clearly separated at aof nitrogen, the collected oxysterols were derivatized for
30 min at 60°
C with Tri-Sil HTP reagent. The derivatized

concentration of 200 ng/mL (Fig. S1B). Table 2 shows therecorded linear regression, respective correlation coeﬃ-

oxysterols were resuspended in 50 μL n-hexane for GC-MS
analysis (Fig. 2).

cients, recovery rates, and mean relative standard deviation(RSD) values of the intra- and inter-day precision for each

GC-MS Analysis

component in the present study. The 22S-hydroxycholesterol, 22R-hydroxycholesterol, and 20S-hydroxycholesterol

The conditions for GC-MS measurements were as follows: capillary column, DB-5MS (30 m × 0.25 mm, 0.25 μm

concentrations were below the limit of detection in plasma.

particle size); carrier gas, helium (1 mL/min); column tem-greater than 0.997 for all the compounds. Recovery rates
ranged from 88.8 to 103.4%. The intra-day precision (RSD)The correlation coeﬃcients of the calibration curves wereperature, 180°
C for 1 min → 20°
C/min → 250°
C → 5°C/

min → 280°
C → 3°
C/min → 300°
C for 12 min; inlet tem-ranged from 2.3 to 11.0% and the inter-day precision (RSD)93June 2023

Table 2.

Medical Mass Spectrometry Vol. 7 No. 1

Validation data for cholesterol, its precursors, plant sterols, oxysterols, and cholestanol in spiked human plasma
Intra-day precision Inter-day precision(RSD, %)(RSD, %)

Calibration curve

Correlationcoeﬃcient

Calibration
range

Recovery(%)

Cholesterol

y＝0.0362x＋0.2049

0.999

̶

3.5

10.7

Cholestanol

y＝0.0138x＋0.0329

0.999

0.3‒3 mg/mL
0.10‒10 μg/mL

99.7±6.2

4.6

3.6

Squalene

y＝0.3384x＋0.0591

0.999

4.3

12.3

Lanosterol

y＝0.0363x＋0.0062

0.999

7.5

14.2

Lathosterol
7-dehydrocholesterol

y＝0.0034x＋0.0040

0.999

0.12‒10 μg/mL 100.1±0.020.12‒10 μg/mL 98.1±1.30.12‒10 μg/mL 101.0±5.5

6.4

2.6

y＝0.0046x＋0.0022

0.999

0.25‒10 μg/mL

97.5±1.6

4.3

2.3

Desmosterol

y＝0.0268x＋0.0158

0.999

0.12‒10 μg/mL 102.0±1.8

2.6

4.6

Campesterol

y＝0.0108x＋0.0222

0.999

3.8

y＝0.0475x＋0.0076

0.999

2.7

12.6

y＝0.0083x＋0.0159

0.999

0.12‒10 μg/mL 102.5±4.3
0.10‒10 μg/mL 88.8±6.0
0.15‒10 μg/mL 100.3±1.0

11.0

Stigmasterol
β-sitosterol

9.2

11.1

Sterols

Cholesterol precursors

Plant sterols

Oxysterols
7α-hydroxycholesterol

y＝0.0322x＋1.3891

0.999

5.0

8.7

y＝0.0246x＋0.5180

0.999

1.72‒200 ng/mL
1.72‒200 ng/mL

97.8±2.47β-hydroxycholesterol
4β-hydroxycholesterol

98.3±0.7

6.2

3.5

y＝0.0152x＋0.3356

0.999

1.72‒200 ng/mL

97.7±3.4

5.6

10.0

Cholesterol-5β,6β-epoxide
Cholesterol-5α,6α-epoxide

y＝0.00111x＋0.0241

0.997

3.1

3.4

y＝0.00084x＋0.02162

0.999

4.7

7.3

24S-hydroxycholesterol

y＝0.0030x＋0.0704

0.999

15‒200 ng/mL 97.0±1.012‒200 ng/mL 102.4±3.85‒200 ng/mL 100.1±0.4

10.0

4.5

25-hydroxycholesterol
7-ketocholesterol

y＝0.0670x＋0.3575

0.999

y＝0.0009x＋0.0552

0.999

27-hydroxycholesterol

y＝0.0017x＋0.1668

0.999

1.72‒200 ng/mL 97.3±0.11.72‒200 ng/mL 100.1±0.91.72‒200 ng/mL 103.4±3.2

7.5

1.5

5.2

13.4

2.3

5.6

Recovery is represented as the mean±standard deviation (n＝3).

ranged from 1.5 to 14.2% (Table 2). This method was

between CTX, sitosterolemia, and FH because cholesterol

highly speciﬁc, sensitive, and reproducible.

could not be measured simultaneously. In contrast to CTX
and sitosterolemia, FH is associated with hypercholesterol-

This method was applied to measure plasma cholesterol,. Therefore, oxysterols are found in cholesterol-richtissues, usually at concentrations several orders of magni3, 9)

its precursors, plant sterols, cholestenol, and oxysterols inemia

six samples. The newly developed method appeared to be12)

1, 13)

congruent with the conventional results , and the accuracy
ranged from 85.0 to 115.5%. The concentrations of choles-

tude lower than cholesterol

large amounts in human plasma, and the normal value for10, 14), whereas oxysterol ischolesterol is 1.6‒2.3 mg/mL

terol and related sterols determined in our study were

present at markedly lower levels, with normal values of10‒12, 15‒17), which is less than 0.01% of choles2‒200 ng/mL

within the same range as those published in the previous
3, 10, 12‒17, 19)

reports. Cholesterol is present in(Table 3).

Several methods for sterol analysis from various biologi-

terol. Thus, to develop a method for the simultaneous deter-

cal matrices have been described, including commercial
3)
3, 14)
10, 17), LC-MS/MS, andassay kits (colorimetric) , HPLC

mination of oxysterols, a large amount of cholesterol must

GC-MS

designed method is superior to the conventional method

be removed before TMS derivatization. Our newly

3, 12, 13, 15, 16, 18)

18). However, to measure cholesterol, its

precursors, plant sterols, cholestanol, and oxysterols, it isin the following respects: this method could be measured

necessary to use diﬀerent pretreatment methods andusing only two ISs, whereas the conventional methods used

18)

18)

developed an analytical method
for the simultaneous determination of four cholesterol pre-ﬁve types of IS

cursors, three plant sterols, and four oxysterols in the

could determine a wider variety of oxysterols to remove thelarge amount of cholesterol with solid-phase extraction atmachines. Matysik et al.

18)

vs. 400 μL

human plasma. This method alone could not distinguish94, the amount of plasma was low (250 μL
by one-round pretreatment), and our method

June 2023

Table 3.

Medical Mass Spectrometry Vol. 7 No. 1

Determination of plasma levels of cholesterol, its precursors, plant sterols, oxysterols, and cholestenol
sample A

Cholesterol (mg/mL)

a 0.847±0.152bCholestanol (μg/mL)

sample B

sample C

1.589±0.232

1.154±0.060 0.955±0.102 1.467±0.317

N.D

N.D

a 1.256±0.045

1.515±0.151b

N.D

sample D

N.D

sample E

N.D

1.473±0.048 2.082±0.057 0.918±0.134
N.D

N.D

N.D

sample F

Mean±S.D.

0.467±0.050 1.080±0.418
N.D

Nomal values

N.D

1.578‒2.300

N.D

1.838±0.999

15)91.6 0.610±0.380

12)10, 14)

N.D

1.054±0.025 1.383±0.395
N.D

Accuracy
(%)

N.D

N.D

N.D

a 0.340±0.038

0.782±0.096

0.417±0.006 0.143±0.015 0.330±0.111

0.181±0.101 0.366±0.224

b 0.369±0.137

0.670±0.108

0.431±0.096 0.262±0.031 0.427±0.043

0.237±0.081 0.399±0.165

a 0.221±0.039

0.126±0.084

0.446±0.129 1.637±0.089 0.147±0.011

0.735±0.563 0.552±0.580

Cholesterol precursors (μg/mL)
Squalene

Lanosterolb
Lathosterol
7-dehydrocholesterol

N.D

N.D

a 0.843±0.082

0.740±0.066

2.140±0.206 0.550±0.021 0.794±0.073

2.013±0.316 1.180±0.674

b 1.068±0.651

0.863±0.420

1.668±0.552 0.566±0.166 0.862±0.105

1.769±0.590 1.113±0.597

a 0.341±0.166

0.296±0.180

0.261±0.008 0.519±0.214 0.591±0.165

0.423±0.163 0.405±0.149bDesmosterol

N.D

N.D

N.D

N.D

N.D

N.D

N.D

N.D

N.D

0.00853‒0.213

16)

N.D

N.D

N.D

a 0.603±0.516

0.845±0.370

0.684±0.099 0.770±0.346 0.998±0.351

0.559±0.263 0.743±0.329

b 0.451±0.233

0.655±0.115

0.759±0.100 0.689±0.230 0.850±0.016

0.527±0.211 0.655±0.199

a 2.084±0.903

4.639±2.589

2.005±0.184 4.574±0.373

3.230±0.326 0.910±0.094 2.907±1.710

b 1.622±0.884

3.553±0.332

1.654±0.688 3.941±1.106

4.783±0.420 0.777±0.313 2.722±1.598

a 0.112±0.008

0.114±0.071

0.129±0.049 0.245±0.119

0.294±0.063 0.113±0.034 0.172±0.057

b 0.109±0.058

0.135±0.019

0.133±0.085 0.214±0.130

0.250±0.010 0.092±0.025 0.156±0.055

a 0.765±0.106

1.095±0.147

1.637±0.240 3.138±2.173

3.148±0.945 0.807±0.485 1.765±1.338

b 0.979±0.546

1.687±0.470

1.596±0.519 3.029±0.976

3.377±0.441 0.602±0.245 1.878±1.147

a 35.95±8.72

50.74±16.64

71.72±28.45 22.05±5.20

33.16±1.92

16.00±3.4738.27±22.49

b 27.80±5.90

43.76±5.76

72.14±3.06

31.22±4.60

17.23±5.00

34.58±21.20

a 23.13±9.25

29.72±1.71

68.54±21.05 27.70±0.97

40.99±17.07 18.92±3.29

34.83±19.70

b 18.25±3.75

36.02±3.1855.64±4.8517.53±5.2333.44±10.75 20.02±9.4530.15±15.06

a 28.48±3.81

14.13±2.8843.19±4.8533.72±7.0845.43±9.37

7.01±1.49

28.66±15.28

N.D

N.D

N.D

N.D

N.D

N.D

104.2 2.056±0.792
N.D

0.039‒0.385

3)

14, 19)N.D
113.4 0.740±0.420

12)106.8 3.160±1.710

12)Plant sterols (μg/mL)
Campesterol

Stigmasterol

β-sitosterol

110.3 0.140±0.110

12)94.0 2.050±1.050

12)

Oxysterols (ng/mL)
7α-hydroxycholesterol

7β-hydroxycholesterol

4β-hydroxycholesterolb

N.D

Cholesterol-5β,6β-epoxide a 46.70±34.68
b

N.D

Cholesterol-5α,6α-epoxide a 35.11±2.57b
24S-hydroxycholesterol

25-hydroxycholesterol

7-ketocholesterola 18.58±1.02

19.37±10.75 118.63±31.98 31.64±5.71
N.D
20.73±5.35

N.D

N.D

61.36±10.66 22.24±3.06

59.33±22.01 44.67±9.78

53.39±37.60

N.D

N.D

N.D

15.27±4.54

27.08±6.03

30.30±16.40

N.D

N.D

N.D

N.D

N.D

N.D

48.68±9.18

37.76±0.78

32.73±1.2110.77±0.83

11.79±1.66

26.72±14.82

b

N.D

N.D

N.D

N.D

N.D

N.D

N.D

a

6.28±0.21

8.34±1.20

9.15±0.14

4.66±0.31

4.23±0.17

3.34±1.04

6.00±2.28

b

N.D

N.D

N.D

N.D

N.D

N.D

N.D

a 52.79±11.96 127.91±20.61 140.44±9.92b27-hydroxycholesterol

N.D

15.33±2.91

63.68±4.68

37.70±11.72 32.32±7.37
N.D

N.D

74.59±46.30

N.D

N.D

N.D

N.D

a 51.44±13.50

68.91±9.45

93.63±5.23

61.63±2.90

137.23±15.21 52.61±10.33 77.58±32.22

b 67.40±4.15111.18±5.93130.70±6.63

58.34±6.55

124.96±8.71

13)

110.7

43±48

115.5

14‒206

N.D

24±11

N.D

26±18

N.D

6±8

N.D

31±4

N.D

2±3

N.D

69±18

10)

17)

13)

13)

10)

13)

10)

N.D
85.0 101.8±22.8

12)54.99±11.27 91.26±33.30
12)

Data are expressed as mean±standard deviation (n＝3). a: Results obtained using the newly developed method; b: Results obtained using a conventional method ; N.D: nodata; Accuracy was determined as the mean percentage of the concentration in plasma measured using the newly developed method with respect to that evaluated using a12)
conventional method .

95June 2023

Medical Mass Spectrometry Vol. 7 No. 1

pretreatment. Furthermore, using our method, the volume2) Müller C, Junker J, Bracher F, Giera M: A gas chromatog-of the organic solvent was reduced by approximately 36%
12)compared to the conventional method . In clinical prac-raphy-mass spectrometry-based whole-cell screening as-

tice, cholesterol is typically measured using the enzyme

sis. Nat Protoc 14: 2546‒2570, 2019.

say for target identiﬁcation in distal cholesterol biosynthe-

method with an autoanalyzer. However, this method may3) Mignarri A, Magni A, Puppo MD, Gallus GN, Björkhem

also detect other related sterols (cholesterol precursors,
20, 21). Therefore, the GC-MS
plant sterols, or oxysterols)I, et al: Evaluation of cholesterol metabolism in cerebro-

method was developed to facilitate speciﬁc, accurate sepa-

2016.

tendinous xanthomatosis. J Inherit Metab Dis 39: 75‒83,4) Endo K, Oyama T, Saiki A, Ban N, Ohira M, et al: Deter-

ration of cholesterol.
This new method of measuring plasma cholesterol, its

mination of serum 7-ketocholesterol concentrations andprecursors, plant sterols, cholestanol, and oxysterols, is a
useful tool in the diagnosis of inherited disorders of choles-

their relationships with coronary multiple risks in diabetes

terol biosynthesis, malformation syndromes, FH, CTX, or
1, 3, 7‒9)and evaluation of drug administrasitosterolemia

5) Wolozin B: Cyp46 (24S-cholesterol hydroxylase): A

10‒12)

tion

mellitus. Diabetes Res Clin Pract 80: 63‒68, 2008.
genetic risk factor for Alzheimer disease. Arch Neurol 60:. This method combines the excellent power of sep-

16‒18, 2003.

aration performed by GC with the high sensitivity of the6) Linseisen J, Wolfram G, Miller AB: Plasma 7β-hydroxy-mass spectrometric detection system operating in the SIM

cholesterol as a possible predictor of lung cancer risk.

mode. In addition, the SIM mode signiﬁcantly increases theCancer Epidemiol Biomarkers Prev 11: 1630‒1637, 2002.

speciﬁcity of the analysis, minimizing the risk of erroneousquantitative determination of sterols due to the co-elution of7) Porter FD, Herman GE: Malformation syndromes causedby disorders of cholesterol synthesis. J Lipid Res 52: 6‒34,

impurities.

2011.
8) Mangili LC, Miname MH, Silva PRS, Bittencourt MS,

Conclusions

Rocha VZ: Achilles tendon xanthomas are associated withWe developed a method to determine cholesterol, its
related sterols, and oxysterols in plasma by a one-roundthe presence and burden of subclinical coronary atherosclerosis in heterozygous familial hypercholesterolemia:

pretreatment using GC-MS. This method has been fully val-

A pilot study. Atherosclerosis 263: 393‒397, 2017.

idated and is speciﬁc, accurate, precise, and reproducible. It
contributes to the diagnosis of inherited disorders of choles-

9) Loh WJ, Watts GF: The Inherited Hypercholesterolemias.

terol biosynthesis, malformation syndromes, FH, CTX, or
sitosterolemia by a one-round pretreatment. Furthermore,

10) Dias IHK, Milic I, Lip GYH, Devitt A, Polidori MC, et al:

this method can be used to evaluate the eﬀects of new

with hypercholesterolaemia. Redox Biol 16: 139‒145, 2018.

Endocrinol Metab Clin North Am 51: 511‒537, 2022.
Simvastatin reduces circulating oxysterol levels in men

drugs used to treat dyslipidemia.

11) Wu AHB: Biomarkers for cholesterol absorption andsynthesis in hyperlipidemic patients: Role for therapeutic

Acknowledgements

selection. Clin Lab Med 34: 157‒66, 2014.

This research was supported by Grants-in-Aid for Sci-

12) Hirayama S, Nakagawa S, Soda S, Kamimura Y, Nishioka

ence Research from the Ministry of Education, Science andCulture of Japan (No. 19K07874, 2019‒2021 to T.M.).

E, et al: Ezetimibe decreases serum oxidized cholesterol
without impairing bile acid synthesis in Japanese hypercholesterolemic patients. Atherosclerosis 230: 48‒51, 2013.

Conﬂict of Interest

13) Dzeletovic S, Breuer O, Lund E, Diczfalusy U: Determi-

The authors declare no conﬂict of interest.

nation of cholesterol oxidation products in human plasma
by isotope dilution-mass spectrometry. Anal Biochem 225:

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