An LC-MS/MS method to quantify acylcarnitine species including isomeric and odd-numbered forms in plasma and tissues

Article abstract of DOI:10.1194/jlr.D061721

Acylcarnitines are intermediates of fatty acid and amino acid oxidation found in tissues and body fluids. They are important diagnostic markers for inherited diseases of peroxisomal and mitochondrial oxidation processes and were recently described as biomarkers of complex diseases like the metabolic syndrome. Quantification of acylcarnitine species can become challenging because various species occur as isomers and/or have very low concentrations. Here we describe a new LC-MS/MS method for quantification of 56 acylcarnitine species with acyl-chain lengths from C2 to C18. Our method includes amino acid-derived positional isomers, like methacrylyl-carnitine (2-M-C3:1-CN) and crotonyl-carnitine (C4:1-CN), and odd-numbered carbon species, like pentadecanoyl-carnitine (C15:0-CN) and heptadecanoyl-carnitine (C17:0-CN), occurring at very low concentrations in plasma and tissues. Method validation in plasma and liver samples showed high sensitivity and excellent accuracy and precision. In an application to samples from streptozotocin-treated diabetic mice, we identified significantly increased concentrations of acylcarnitines derived from branched-chain amino acid degradation and of odd-numbered straight-chain species, recently proposed as potential biomarkers for the metabolic syndrome. In conclusion, the LC-MS/MS method presented here allows robust quantification of isomeric acylcarnitine species and extends the palette of acylcarnitines with diagnostic potential derived from fatty acid and amino acid metabolism.

Full text of DOI:10.1194/jlr.D061721

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methods
An LC-MS/MS method to quantify acylcarnitine species
including isomeric and odd-numbered forms in plasma
and tissues
Pieter Giesbertz, Josef Ecker, Alexander Haag, Britta Spanier, and Hannelore Daniel1
Nutritional Physiology, Technische Universität München, 85350 Freising, Germany; and ZIEL Institute for
Food and Health, 85350 Freising, Germany
Abstract Acylcarnitines are intermediates of fatty acid and
amino acid oxidation found in tissues and body fluids. They
are important diagnostic markers for inherited diseases of
peroxisomal and mitochondrial oxidation processes and
were recently described as biomarkers of complex diseases
like the metabolic syndrome. Quantification of acylcarni-
tine species can become challenging because various spe-
cies occur as isomers and/or have very low concentrations.
Here we describe a new LC-MS/MS method for quantifi-
cation of 56 acylcarnitine species with acyl-chain lengths
from C2 to C18. Our method includes amino acid-derived
positional isomers, like methacrylyl-carnitine (2-M-C3:1-CN)
and crotonyl-carnitine (C4:1-CN), and odd-numbered car-
bon species, like pentadecanoyl-carnitine (C15:0-CN) and
heptadecanoyl-carnitine (C17:0-CN), occurring at very low
concentrations in plasma and tissues. Method validation in
plasma and liver samples showed high sensitivity and excel-
lent accuracy and precision. In an application to samples
from streptozotocin-treated diabetic mice, we identified
significantly increased concentrations of acylcarnitines de-
rived from branched-chain amino acid degradation and of
odd-numbered straight-chain species, recently proposed
as potential biomarkers for the metabolic syndrome. In
conclusion, the LC-MS/MS method presented here allows
robust quantification of isomeric acylcarnitine species and
extends the palette of acylcarnitines with diagnostic poten-
tial derived from fatty acid and amino acid metabolism.—
Giesbertz, P., J. Ecker, A. Haag, B. Spanier, and H. Daniel.
An LC-MS/MS method to quantify acylcarnitine species in-
cluding isomeric and odd-numbered forms in plasma and
tissues. J. Lipid Res. 2015. 56: 2029–2039.
transport of activated fatty acids (acyl-CoAs) from the cyto-
sol across the inner mitochondrial membrane into the
matrix where fatty acid oxidation takes place. It has now
become clear that a net efflux of acylcarnitine species
from the mitochondria into the cytosol and ultimately into
plasma is particularly important in situations of impaired
fatty acid oxidation to prevent accumulation of potentially
toxic acyl-CoA intermediates in the mitochondrion (2, 3).
How acylcarnitines are released into the extracellular
space is not known, but changes in plasma and/or urinary
acylcarnitine profiles are used to detect disorders in fatty
acid and amino acid oxidation (4). More recently, altera-
tions in plasma acylcarnitine profiles were described as
strongly associated with obesity and type 2 diabetes (5–7).
A broad spectrum of short-, medium-, and long-chain acyl-
carnitine species is generated from multiple metabolic
routes including various fatty acid and amino acid oxida-
tion pathways. A comprehensive quantification that covers
as many as possible of these entities is therefore not only
important for proper clinical diagnosis of inherited enzy-
matic impairments, but also for basic studies of complex
diseases such as the metabolic syndrome.
Acylcarnitine profiles are commonly established by
direct infusion ESI-MS/MS. This technology, however,
does not allow the discrimination of isomeric acylcarni-
tine species. Furthermore, isobaric matrix interferences
can lead to an overestimation of metabolite concentra-
tions producing false positive results, in particular, for
compounds with low abundance (8). Although LC-MS/
MS methods have been developed for successful separa-
tion of isomeric compounds (9–12), the identification of
the exact compound in the chromatogram often remains
Supplementary key words diabetes • fatty acid/oxidation • mitochon-
dria • liver • diagnostic tools • liquid chromatography-tandem mass
spectroscopy
Acylcarnitines are intermediates in fatty acid and amino
acid breakdown generated from the conversion of acyl-
CoA species by the action of carnitine acyltransferases (1).
The formation of carnitine conjugates is crucial for the
Abbreviations: CN, carnitine; CV, coefficient of variation; HFBA,
heptafluorobutyric acid; IS, internal standard; LOD, limit of detection;
LOQ, limit of quantitation; MRM, multiple reaction monitoring; STZ,
streptozotocin.
1 To whom correspondence should be addressed.
e-mail: hannelore.daniel@tum.de
Manuscript received 8 July 2015 and in revised form 24 July 2015.
Published, JLR Papers in Press, August 3, 2015
DOI 10.1194/jlr.D061721
The online version of this article (available at http://www.jlr.org)
contains a supplement.
Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.
This article is available online at http://www.jlr.org
Journal of Lipid Research Volume 56, 2015
2029

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acid and for (S)-␤-hydroxyisobutyric acid, this protocol gener-
ated a racemic mixture of both (S)- and (R)- stereoisomers.
elusive. Additionally, the abundance of many species in
biological samples is often too low for robust detection
and quantification.
Sample preparation and derivatization
Here we describe a robust and highly sensitive LC-MS/
MS method for comprehensive quantification of an array
of acylcarnitine species with a special focus on amino acid-
derived intermediates, which often occur at very low con-
centrations and in isomeric forms. Because alterations in
plasma odd-numbered fatty acids were recently shown to
associate with impaired glucose tolerance and diabetes
(13), we also focused on quantification of the correspond-
ing odd-numbered acylcarnitines. The method was validated
and applied to plasma and liver samples from streptozoto-
cin (STZ)-induced insulin-deficient mice, which serve as a
model of type I diabetes.
Plasma (10 ␮l) was dissolved in 100 ␮l ice-cold methanol con-
taining the IS mixture under ultra-sonication to prevent sample
aggregation. Dissolved samples were evaporated to dryness in a
vacuum concentrator. Tissue samples were first grounded in liq-
uid nitrogen using pestle and mortar. Forty milligrams of tissue
were then extracted using 1,800 ␮l 100% ice-cold methanol and
centrifuged at 10,000 g, 4°C for 10 min. IS was added to 200 ␮l of
the supernatant before vacuum-drying the samples.
Acylcarnitines were derivatized to their butyl esters as described
by Gucciardi et al. (9). Briefly, 100 ␮l n-butanol containing 5%
v/v acetyl chloride was added to the dried samples, incubated at
60°C for 20 min at 800 rpm (Eppendorf Thermomixer Comfort;
Eppendorf, Hamburg, Germany) and subsequently evaporated
to dryness. Samples were reconstituted in 100 ␮l (for plasma
samples) or 200 ␮l (for tissue samples) methanol/water and
transferred to glass vials.
MATERIALS AND METHODS
LC-MS/MS
Chemicals, solutions, reference substances, and internal
standards
TheanalysiswasperformedonatriplequadrupoleQTRAP5500
LC-MS/MS system (AB Sciex, Framingham, MA), equipped with
a 1200 series binary pump, a degasser, and a column oven (Agilent,
Santa Clara, CA) connected to a HTC pal autosampler (CTC
Analytics, Zwingen, Switzerland). A Turbo V ion spray source op-
erating in positive ESI mode was used for ionization. The source
settings were: ion spray voltage, 5,500 V; heater temperature,
600°C; source gas 1, 50 psi; source gas 2, 50 psi; curtain gas, 40
psi; and collision gas (CAD), medium.
Chromatographic separation was achieved on a Zorbax Eclipse
XDB-C18 column (length 150 mm, internal diameter 3.0 mm,
particle size 3.5 ␮m; Agilent) at a column temperature of 50°C.
Eluent A consisted of 0.1% formic acid, 2.5 mM ammonium ace-
tate, and 0.005% HFBA in water. Eluent B consisted of 0.1% for-
mic acid, 2.5 mM ammonium acetate, and 0.005% HFBA in
acetonitrile. Gradient elution was performed with the following
program: 100% A (0.5 ml/min) for 0.5 min, a linear decrease to
65% A (0.5 ml/min) for 2.5 min, hold for 3 min, a linear de-
crease to 40% A (0.5 ml/min) for 3.7 min, a linear decrease to
5% A (0.5 ml/min) for 1 min. Elution was then carried out at
100% B (1 ml/min) for 0.5 min, hold for 7.3 min (1.5 ml/min).
Re-equilibration was finally performed at 100% A (0.5 m/min)
for 4 min. The complete running time of the program was 22
min. Analytes were measured in scheduled multiple reaction
monitoring (MRM) (total scan time, 0.5 s; scan time window, 0.5
min). Quadrupoles were working at unit resolution.
Ammonium acetate, n-butanol (99.7%), thionyl chloride, car-
nitine hydrochloride, formic acid (LC-MS grade), heptafluoro-
butyric acid (HFBA) (у99.5%), acetyl chloride, and acetonitrile
(LC-MSgrade)werepurchasedfromSigma-Aldrich(Taufkirchen,
Germany). Water (LC-MS grade) and diethyl ether (Baker ana-
lyzed) were obtained from J. T. Baker Chemicals (Center Valley,
PA). Methanol (LC-MS grade) and trichloroacetic acid were pur-
chased from Merck (Darmstadt, Germany). The internal standard
(IS) mixture containing d9-carnitine (0.785 pmol/␮l), d3-acetyl-
carnitine (0.327 pmol/␮l), d3-propionyl-carnitine (0.086 pmol/␮l),
d3-butyryl-carnitine (0.043 pmol/␮l), d9-isovaleryl-carnitine
(0.039 pmol/␮l), d3-hexanoyl-carnitine (0.046 pmol/␮l), d3-oc-
tanoyl-carnitine (0.042 pmol/␮l), d3-decanoyl-carnitine (0.046
pmol/␮l), d3-dodecanoyl-carnitine (0.043 pmol/␮l), d3-tetradec-
anoyl-carnitine (0.041 pmol/␮l), d3-hexadecanoyl-carnitine (0.082
pmol/␮l), and d3-octadecanoyl-carnitine (0.074 pmol/␮l) was
obtained from ChromSystems (München, Germany). The free
fatty acids used for synthesis of acylcarnitines (crotonoic acid,
methacrylic acid, tiglic acid, dimethylacrylic acid, isobutyric acid,
2-methylbutyric acid, (S)-3-hydroxybutyric acid, (R)-3-hydroxybu-
tyric acid, sodium-(S)-␤-hydroxyisobutyric acid, 3-hydroxyisovale-
ric acid, trans-2-methyl-butenoic acid, heptanoic acid, nonanoic
acid, undecanoic acid, tridecanoic acid, pentadecanoic acid,
heptadecanoic acid, and adipic acid) were obtained from Sigma-
Aldrich. Valeryl-carnitine was acquired from LGC-Standards (Wesel,
Germany), 15-methylhexadecanoic acid was obtained from Laro-
dan (Solna, Sweden), and 3-methylglutaryl-carnitine was pur-
chased at Avanti Polar Lipids (Alabaster, AL).
Calibration and quantification
Calibration was achieved by spiking plasma and liver samples
with different quantities of acylcarnitine standards. A ten-point
calibration was performed by addition of increasing amounts of
each standard and IS, as described in the sample preparation sec-
tion. Calibration curves were constructed and fitted by linear re-
gression without weighting. Data analysis was done using Analyst
1.5® software (AB Sciex).
Synthesis of reference substances
O-acylated acylcarnitines were synthesized from carnitine chlo-
ride and free fatty acids using a modified protocol from Ziegler
et al. (14). Briefly, the free fatty acid was incubated with thionyl
chloride (15 mg free fatty acid per microliter thionyl chloride)
under continuous shaking for 4 h at 70°C to generate fatty acyl
chlorides. Carnitine hydrochloride dissolved in trichloroacetic
acid was added to the acyl chloride and incubated at 45°C for 18 h.
After cooling, the product was precipitated and washed three
times in cold diethyl ether. Sodium-(s)-␤-hydroxyisobutyric acid
was first treated with hydrochloric acid to produce the free fatty
acid, followed by evaporation of water at 90°C for 30 min, before
incubation with thionyl chloride. For (S)- and (R)-␤-hydroxybutyric
Animal experiment and sample collection
C57BL6/N mice (n = 10), at an age of 12 weeks, were injected
intraperitoneally with a single dose of STZ (180 mg/kg body
weight dissolved in 0.1 M citric acid buffer) and mice (n = 10)
injected intraperitoneally with citric acid buffer served as con-
trols. All mice were fed a standard chow diet (ssniff V1534-0 R/M-H)
and were not fasted prior to sampling of blood and organs. Body
2030
Journal of Lipid Research Volume 56, 2015

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weight and blood glucose levels were measured daily using tail-
spectra were acquired. Butylation of acylcarnitines (espe-
cially of dicarboxylic species) increased the ionization ef-
ficiency (data not shown). Representative fragmentation
patterns for valeryl-carnitine (C5:0-CN; monocarboxylic)
and 3-methylglutaryl-carnitine (C5:0-M-DC-CN; dicarbox-
ylic) are shown in Fig. 1. Fragmentation patterns of acyl-
carnitines were reported to have a prominent fragment
ion at m/z 85 in common (15). This fragment ion displayed
the highest intensity in our measurements and was thus
used for quantification. It is proposed to be formed due to
loss of the butyl group(s) (m/z 56), the acyl chain [m/z 102
and m/z 146 for valeryl-carnitine (C5:0-CN) and 3-methyl-
glutaryl-carnitine (C5:0-M-DC-CN), respectively], and the
trimethylamine fragment (m/z 59), as initially described by
Chace et al. (15) in 1997. A compilation of all acylcarni-
tine species covered by our method, including the corre-
sponding MS parameters, is shown in Table 1.
vein puncture. After 5 days, mice were anesthetized with isoflu-
rane. Blood was collected into EDTA-coated tubes and was
centrifuged for 10 min at 1,200 g and 4°C. Plasma was separated
and snap-frozen in liquid nitrogen. Mice were euthanized and
liver tissue was collected and immediately snap-frozen in liquid
nitrogen. All samples were stored at Ϫ80°C until the day of mea-
surement. The research was conducted in conformity with the
Public Health Service policy on humane care and use of labora-
tory animals. All experiments were performed according to
the German guidelines for animal care and were approved by the
state of Bavaria (Regierung von Oberbayern) ethics committee
(reference number 55.2.1-54-2532-22-11).
Statistical analysis
Levels of significance between metabolite concentrations in
STZ-treated and control mice were assessed using two-sided inde-
pendent Student’s t-tests.
Chromatographic separation and identification of
isomeric acylcarnitine species
RESULTS
Sample extraction and derivatization
Because all acylcarnitines form a similar product ion
(m/z 85) and many compounds are isobaric or occur as
positional isomers, appropriate chromatographic separa-
tion is essential. Chromatographic separation was estab-
lished using a C18-reversed phase HPLC column (length,
15 cm; internal diameter, 3.0 mm; particle size, 3.5 ␮m).
We favored an HPLC column over an ultra-performance
LC column, because larger sample volumes of complex
matrices were injected. Peaks were well shaped, sharp
(ف0.1 min; Fig. 2), and well separated. A coelution of
analyte and IS was achieved, which is important to com-
pensate for matrix effects and varying ionization efficien-
cies during gradient elution (16). The addition of HFBA
to the eluents as an ion-pairing reagent (17) improved peak
Extraction of acylcarnitine species from plasma and tis-
sue samples was achieved using methanol and analytes
were derivatized to their butyl esters. Because dicarboxylic
acylcarnitines were derivatized at both carboxyl groups
(see Fig. 1), we could discriminate various isobaric acylcar-
nitines, which have different masses as corresponding butyl
esters [e.g., hydroxybutyryl-carnitine (C4-OH-CN, nonder-
ivatized m/z 248, butylated m/z 304) and malonyl-carnitine
(C3:0-DC-CN, nonderivatized m/z 248, butylated m/z 360)].
Acylcarnitine fragmentation
Mass spectrometric analysis of acylcarnitine species was
performed using ESI in positive mode and product ion
Fig. 1. Product ion spectra and proposed fragmentation patterns for valeryl-carnitine (C5:0-CN) (A) and
3-methylglutaryl-carnitine (C5-M-DC-CN) (B).
An LC-MS/MS method to quantify acylcarnitine isomers
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TABLE 1. MS parameters and retention times
Retention
Time (min)
MRM (m/z)
IS
IS MRM (m/z)
DP (V)
EP (V)
CE (V)
CXP (V)
Free carnitine
C0
218.1 → 85.1
290.1 → 85.1
304.1 → 85.1
304.1 → 85.1
d9-C0
d3-C3
d3-C4
d3-C4
d3-C4
d3-C2
d9-C5
d3-C3
d3-C6
d3-C4
d3-C6
d3-C4
d3-C4
d3-C4
d9-C5
d9-C5
d9-C5
d9-C5
d9-C5
227.5 → 85.1
277.1 → 85.1
291.4 → 85.1
291.4 → 85.1
291.4 → 85.1
263.2 → 85.1
311 → 85.1
5.9
6.1
6.2
6.3
6.4
6.4
6.5
6.9
7.3
7.3
7.5
7.5
7.8
8
8.2
8.4
9.5
61
53
55
55
55
41
58
46
61
52
61
52
46
46
55
55
46
46
46
56
55
56
56
58
61
60
63
63
60
66
66
68
66
56
65
65
78
73
87
80
80
83
86
87
87
92
84
85
85
89
84
89
90
90
86
96
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
23
30
32
32
32
18
33
29
35
29
35
29
29
29
31
31
29
29
29
27
32
33
27
33
35
35
37
37
30
39
33
40
39
37
40
40
47
44
53
45
45
51
45
53
53
57
51
48
48
54
51
55
57
57
45
63
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
Hydroxypropionyl-carnitine
Hydroxybutyryl-carnitine a
Hydroxybutyryl-carnitine b
3-Hydroxyisobutyryl-carnitine
Acetyl-carnitine
C3-OH
C4-OH a
C4-OH b
2-M-C3-OH 304.1 → 85.1
C2
C5-OH
C3:0
C6-OH a
C4:1
C6-OH b
2-M-C3:1
2-M-C3:0
C4:0
2-M-C4:1
3-M-C4:1
2-M-C4:0
3-M-C4:0
C5:0
C4:1-DC
C3-DC
C4-DC
C6:0
C3-DC-M
C5-DC
C5:1-DC
C6-DC
C5-M-DC
C7:0
260.5 → 85.1
318.1 → 85.1
274.4 → 85.1
332.2 → 85.1
286.1 → 85.1
332.2 → 85.1
286.1 → 85.1
288.2 → 85.1
288.2 → 85.1
300.2 → 85.1
300.2 → 85.1
302.1 → 85.1
302.1 → 85.1
302.1 → 85.1
Hydroxyvaleryl-carnitine
Propionyl-carnitine
Hydroxyhexanoyl-carnitine a
Crotonyl-carnitine
Hydroxyhexanoyl-carnitine b
Methacrylyl-carnitine
Isobutyryl-carnitine
Butyryl-carnitine
Tiglyl-carnitine
3-Methylcrotonyl-carnitine
2-Methyl-butyryl-carnitine
Isovaleryl-carnitine
Valeryl-carnitine
Fumaryl-carnitine
Malonyl-carnitine
Succinyl-carnitine
Hexanoyl-carnitine
Methyl-malonyl-carnitine
Glutaryl-carnitine
Glutaconyl-carnitine
Adipyl-carnitine
277.1 → 85.1
319.2 → 85.1
291.4 → 85.1
319.2 → 85.1
291.4 → 85.1
291.4 → 85.1
291.4 → 85.1
311 → 85.1
311 → 85.1
311 → 85.1
311 → 85.1
9.6
9.7
9.9
311 → 85.1
372.5 → 85.1 d6-C5-DC 394.2 → 85.1
360.2 → 85.1 d6-C5-DC 394.2 → 85.1
374.2 → 85.1 d6-C5-DC 394.2 → 85.1
10.3
10.8
10.9
11.1
11.1
11.2
11.3
11.4
11.5
11.7
11.9
12.1
12.1
12.3
12.4
12.5
12.6
12.7
12.8
13
13.1
13.1
13.4
13.4
13.5
13.5
13.6
13.6
13.7
13.7
14.3
14.3
14.7
14.8
15.6
15.6
316.4 → 85.1
d3-C6
319.2 → 85.1
374.2 → 85.1 d6-C5-DC 394.2 → 85.1
388.3 → 85.1 d6-C5-DC 394.2 → 85.1
386.3 → 85.1 d6-C5-DC 394.2 → 85.1
402.3 → 85.1 d6-C5-DC 394.2 → 85.1
402.3 → 85.1 d6-C5-DC 394.2 → 85.1
Methylglutaryl-carnitine
Heptanoyl-carnitine
Pimelyl-carnitine
Octanoyl-carnitine
Decenoyl-carnitine
Nonanoyl-carnitine
Decanoyl-carnitine
330.2 → 85.1
d3-C6
319.2 → 85.1
C7-DC
C8:0
C10:1
C9:0
C10:0
Iso-C11:0
C11:0
C14:2
416.3 → 85.1 d6-C5-DC 394.2 → 85.1
344.1 → 85.1
370.2 → 85.1
358.2 → 85.1
372.2 → 85.1
386.3 → 85.1
386.3 → 85.1
424.3 → 85.1
400.3 → 85.1
470.3 → 85.1
414.3 → 85.1
414.3 → 85.1
452.3 → 85.1
428.3 → 85.1
472.3 → 85.1
472.3 → 85.1
498.4 → 85.1
454.3 → 85.1
442.3 → 85.1
442.3 → 85.1
480.3 → 85.1
456.5 → 85.1
482.3 → 85.1
470.4 → 85.1
470.4 → 85.1
d3-C8
d3-C10
d3-C8
347.4 → 85.1
375.2 → 85.1
347.4 → 85.1
375.2 → 85.1
375.2 → 85.1
375.2 → 85.1
431.2 → 85.1
403.2 → 85.1
459.2 → 85.1
403.2 → 85.1
403.2 → 85.1
459.2 → 85.1
431.2 → 85.1
459.2 → 85.1
459.2 → 85.1
487.5 → 85.1
459.2 → 85.1
431.2 → 85.1
431.2 → 85.1
487.5 → 85.1
459.2 → 85.1
487.5 → 85.1
459.2 → 85.1
459.2 → 85.1
d3-C10
d3-C10
d3-C10
d3-C14
d3-C12
d3-C16
d3-C12
d3-C12
d3-C16
d3-C14
d3-C16
d3-C16
d3-C18
d3-C16
d3-C14
d3-C14
d3-C18
d3-C16
d3-C18
d3-C16
d3-C16
Iso-undecanoyl-carnitine
Undecanoyl-carnitine
Tetradecadienyl-carnitine
Dodecanoyl-carnitine
Hydroxyhexadecenyl-carnitine
Isotridecanoyl-carnitine
Tridecanoyl-carnitine
Hexadecadienyl-carnitine
Tetradecanoyl-carnitine
Hydroxyhexadecanoyl-carnitine a
Hydroxyhexadecanoyl-carnitine b
Hydroxyoctadecenyl-carnitine
Hexadecenyl-carnitine
Isopentadecanoyl-carnitine
Pentadecanoyl-carnitine
Octadecadienyl-carnitine
Hexadecanoyl-carnitine
Octadecenyl-carnitine
Isoheptadecanoyl-carnitine
Heptadecanoyl-carnitine
Dodecadioyl-carnitine
Octadecanoyl-carnitine
C12:0
C16:1-OH
Iso-C13:0
C13:0
C16:2
C14:0
C16-OH a
C16-OH b
C18:1-OH
C16:1
iso-C15:0
C15:0
C18:2
C16:0
C18:1
Iso-C17:0
C17:0
C12-DC
C18:0
486.4 → 85.1 d6-C5-DC 394.2 → 85.1
484.4 → 85.1 487.5 → 85.1
d3-C18
DP, declustering potential; EP, entrance potential; CE, collision energy; CXP, collision cell exit potential.
separation and peak sharpness. Addition of ammonium
acetate enhanced ionization efficiency. Figure 2A shows
a typical chromatogram (total ion count) derived from
a liver sample containing acylcarnitines ranging from
0 to 18 carbon atoms. Retention times increased with
the number of carbons and decreased with the number of
double bonds. Because >50 mass transitions were required
to quantify all metabolites, data were acquired in the sched-
uled MRM mode to yield more and shorter scan cycles per
peak, to facilitate peak integration and data analysis.
Various acylcarnitine isomer species were expected to
result from known amino acid and fatty acid breakdown
pathways (18). Indeed, chromatographic separation gen-
erated multiple peaks for various m/z transitions [e.g.,
2-methylbutyryl-carnitine (2-M-C4:0-CN), 3-methylbutyryl-
carnitine (3-M-C4:0-CN), and valeryl-carnitine (C5:0-CN)].
For peak identification, matrix samples were spiked with
individual acylcarnitine reference compounds as shown
in Fig. 2B–D. In general, for saturated acylcarnitines,
branched-chain forms elute earlier than straight-chain
2032
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Fig. 2. Total ion count (TIC) chromatogram (A) and MRM chromatograms of exemplified spiking experi-
ments (B–D).
forms [e.g., isobutyryl-carnitine (2-M-C3:0-CN) < butyryl-
carnitine (C4:0-CN)]. This was also true for odd-num-
bered acylcarnitines, for which two peaks were generally
found (see Fig. 2D). In contrast, for monounsaturated and
dicarboxylic acylcarnitines, branched-chain forms eluted
later [e.g., crotonyl-carnitine (C4:1-CN) < methacrylyl-
carnitine (2-M-C3:1-CN), see Fig. 2B, C]. Furthermore,
hydroxy-acylcarnitines [hydroxybutyryl-carnitine (C4-OH-
CN), hydroxyhexanoyl-carnitine (C6-OH-CN), hydroxy-
hexadecanoyl-carnitine (C16-OH-CN)], displayed two peaks.
This was in accordance with previous reports suggesting
the presence of S- and R-stereoisomers (9, 11, 19).
corresponding IS, analyte-IS pairs were selected accord-
ing to the closest retention times and the best accuracies
for these analyte-IS allocations [e.g., nonanoyl-carnitine
(C9:0-CN) to d3-octanoyl-carnitine (d3-C8:0-CN)]. For all
analytes and matrices, calibration curves were linear and
correlation coefficients (r2) were greater than 0.99
(Table 2). Limits of detection (LODs) were determined
as the signal-to-noise ratio of three, and limits of quanti-
tation (LOQs) were calculated as the triple fold of the
LOD and were sufficient enough for quantification
of acylcarnitine species in plasma and liver samples
(Table 2).
Calibration and quantification
Method validation and sample stabilities
For quantification of metabolite concentrations and
to compensate for variation in sample preparation and
ionization efficiency, an acylcarnitine standard mixture
containing 13 deuterium-labeled acylcarnitine species was
added to the samples (Table 2). Calibration lines were
generated by addition of different concentrations of acyl-
carnitine species to a pool of liver and plasma samples.
The ratio between analyte and IS was used for quantifica-
tion (“stable isotope dilution”). For those analytes without
The accuracy of the method was tested in three liver and
three plasma samples by spiking with acylcarnitine stan-
dards in concentrations covering the whole calibration
range. Accuracies between 81 and 108% were found for
the liver matrix and between 89 and 120% for the plasma
matrix (Table 3), except for the lowest levels of free carnitine
in liver tissues for which concentrations were overestimated.
Method reproducibility was examined by determining
intra- and inter-day precisions in pools of plasma and liver
An LC-MS/MS method to quantify acylcarnitine isomers
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TABLE 2. Calibration data
Calibration Data (Plasma Matrix)
Calibration IS
Calibration Data (Liver Matrix)
Calibration Range IS Added
LOD
LOQ
LOD
LOQ
Correlation (pmol/mg (pmol/mg
Range (pmol/␮l) (pmol/␮l) Slope
R2 (pmol/␮l) (pmol/␮l) (pmol/mg w.w.) (pmol/mg w.w.) Slope Coefficient (R2)
w.w.)
w.w.)
C0
1.5–500
0.375–125
0.075–25
0.075–25
0.075–25
0.075–25
0.075–25
0.075–25
0.075–25
0.075–25
0.15–50
7.70
3.21
0.84
0.42
0.42
0.38
0.38
0.45
0.41
0.45
0.42
0.40
0.80
0.73
0.28 0.999 0.089
0.34 0.999 0.038
1.37 0.999 0.008
3.40 0.999 0.008
3.49 0.999 0.007
5.67 0.999 0.006
5.95 0.999 0.007
3.18 0.999 0.013
2.27 0.997 0.006
2.43 0.993 0.009
2.45 0.992 0.009
2.56 0.996 0.009
1.80 0.998 0.002
1.31 0.994 0.021
0.266
0.113
0.024
0.023
0.021
0.018
0.021
0.038
0.020
0.028
0.027
0.028
0.007
0.065
1.5–1,000
0.375–250
0.075–50
0.075–50
0.075–50
0.075–50
0.075–50
0.075–50
0.075–50
0.075–50
0.15–100
0.15–100
0.15–100
0.15–100
180.55
75.21
19.78
9.89
9.89
8.97
8.97
10.58
9.66
10.58
9.89
9.43
0.0088
0.0099
0.0389
0.098
0.101
0.159
0.158
0.098
0.071
0.083
0.093
0.104
0.064
0.049
0.990
0.999
0.999
0.999
0.999
0.999
0.999
0.999
0.999
0.999
0.999
0.999
0.999
0.999
0.275
0.074
0.003
0.008
0.004
0.003
0.003
0.008
0.002
0.005
0.004
0.003
0.002
0.021
0.824
0.222
0.008
0.011
0.012
0.009
0.009
0.024
0.006
0.014
0.012
0.009
0.005
0.063
C2
C3
C4
2-M-C3
C5
3-M-C4
C6
C8
C10
C12
C14
C16
C18
0.15–50
0.15–50
0.15–50
18.86
17.02
Calibration lines were generated by plotting the area ratios of analyte/IS against the spiked concentrations. Concentrations in liver are given
per milligram of liver wet weight. w.w., wet weight.
samples (Table 4). With a few exceptions, intra- and inter-
day coefficients of variation (CVs) for liver and plasma
samples were below 10% for high-abundant acylcarnitines
(>0.01 pmol/mg tissue; >0.01 pmol/␮l plasma) and be-
tween 10 and 20% for low-abundant acylcarnitines. Re-
coveries were calculated as the difference between area
ratios of prespiked samples (addition of standards before
extraction) and postspiked samples (addition of standards
to the methanol extract). Recoveries between 59 and
99% were found (supplementary Table 1). Matrix effects
were determined by comparing the peak areas of acylcar-
nitine species spiked in matrix samples (three plasma and
three liver samples) and corresponding amounts dis-
solved in methanol at two levels (supplementary Tables 2, 3).
The area ratios of spiked acylcarnitine species in plasma
samples ranged between 110 and 120% compared with
nonmatrix samples. Area ratios of spiked acylcarnitine
species in liver were between 85 and 122% compared
with nonmatrix samples, with a few exceptions for short-
chain species.
To assess sample stability in plasma, samples were stored
at room temperature for 1 h before extraction and peak
areas were compared with samples that were directly ex-
tracted. Acylcarnitines in plasma remained stable over 1 h
at room temperature. For liver tissue, thawing of samples
in three freeze-thaw cycles strongly affected the abun-
dance of various analytes, but postpreparative storage of
plasma and liver samples at Ϫ20°C showed a high stability
of most analytes. See supplementary Table 4 for a sum-
mary on plasma and liver sample stabilities.
We found that concentration ranges of acylcarnitine
species in plasma and liver were in good agreement with
levels reported previously (21), with highest concentra-
tions for saturated short-chain species [acetyl-carnitine
(C2:0-CN, ف20%), propionyl-carnitine (C3:0-CN, ف5%),
and butyryl-carnitine (C4:0-CN, ف2%)] and lowest concen-
trations for odd-numbered medium- and long-chain species
[undecanoyl-carnitine (C11:0-CN), tridecanoyl-carnitine
(C13:0-CN), and pentadecanoyl-carnitine (C15:0-CN)].
Whereas almost all acylcarnitine species could be found in
liver samples, the concentrations in plasma were generally
far lower and a number of species (especially monounsatu-
rated species like crotonyl- and 3-methyl-crotonyl-carnitine)
could not be detected.
Comparison of acylcarnitine concentrations between
diabetic and healthy mice revealed strong increases in the
concentrations of various branched-chain amino acid-de-
rived compounds in plasma and liver of diabetic mice
(Fig. 3A–D). These included leucine-derived isovaleryl-
carnitine (3-M-C4-CN) and 3-methylcrotonyl-carnitine
(3-M-C4:1-CN) and isoleucine-derived 2-methylbutyryl-
carnitine (2-M-C4-CN) and 2-methylcrotonyl-carnitine
(2-M-C4:1-CN), as well as propionyl-carnitine (C3-CN) and
methylmalonyl-carnitine (C3-M-DC-CN). In contrast, 3-hy-
droxyisobutyryl-carnitine (2-M-C3-OH-CN), which is derived
from valine breakdown, showed a marked decrease, both
in plasma and liver. Furthermore, elevated levels of various
straight-chain odd-numbered acylcarnitines were observed
in diabetic mice, most strongly in liver samples (Fig. 3E, F),
whereas decreased levels were found for hexadecenoyl-carni-
tine (C16:1-CN) in plasma and liver and tetradecanoyl-carni-
tine (C14:0-CN) in liver. Supplementary Table 6 provides
concentrations of all acylcarnitine species quantified in
liver and plasma of healthy and diabetic mice in our study.
Method application
The developed method was applied to plasma and liver
samples from STZ-treated insulin-deficient and healthy
control mice. STZ causes pancreatic ␤-cell destruction
resulting in insulin-deficient type I diabetes (20). STZ-
treated animals showed very low circulating insulin levels
accompanied by a dramatic weight loss and a severe in-
crease in blood glucose levels 5 days after treatment (see
supplementary Table 5).
DISCUSSION
Acylcarnitines have recently gained considerable inter-
est as markers for impairments in fatty acid and amino
2034
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TABLE 3. Accuracies
ion at m/z 85 for all analytes, which was described to be a
specific product ion of acylcarnitine fragmentation (15).
LC with a C18-reversed phase HPLC column allowed the
chromatographic separation of isomeric acylcarnitine
species, and by spiking with reference compounds, we
could subsequently assign the individual isomers to the
respective peaks. Proper chromatographic separation is
mandatory because positional isomers have similar mass
transitions [e.g., crotonyl-carnitine (C4:1-CN) and meth-
acrylyl-carnitine (2-M-C3:1-CN), m/z 286 → m/z 85]. In
addition, quantification of acylcarnitine concentrations in
a direct infusion approach is challenged by potential inter-
fering matrix components which lead to an overestima-
tion of analyte concentrations (9). Although a separation
of underivatized acylcarnitine isomers has been described
(11), we applied a butylation to achieve an enhanced
sensitivity, particularly for dicarboxylic acylcarnitines, as
described previously (24). To further improve chromato-
graphic separation and to enhance peak shapes, the ion
pairing agent, HFBA, was added to the eluents in very low
concentrations (0.005%). HFBA was favored over the
stronger trifluoroacetic acid, which is more commonly
used (9), because trifluoroacetic acid causes very strong
ion suppression (25). We could achieve baseline separa-
tion of all acylcarnitine species including positional iso-
mers, and all peaks were well shaped. The total running
time of 22 min is in the range of other reported methods
(9–11).
Calibration was performed by stable isotope dilution.
A coelution of standards and available ISs was achieved,
which is important for compensation of matrix effects
and differences in ionization efficiency (16). LODs and
LOQs were sufficient to determine analytes in plasma
and/or liver (supplementary Table 6). In addition,
we observed that matrix effects of liver tissue are compa-
rable to other tissue types such as muscle, heart, or kid-
ney tissue, and thus suggest that the method can be
applied to other tissues to determine analyte concentra-
tions in these matrices (data not shown). Importantly,
extraction of increased tissue quantities (>40 mg) does
not improve method performance, because this leads
to increased noise and reduced signal-to-noise ratios. In
general, for comparison of analyte concentrations be-
tween samples, it is recommended to extract similar
quantities of tissue to reduce variation resulting from ma-
trix effects.
Liver Spiked
(pmol/mg w.w.) Accuracy (%)
Plasma Spiked
(pmol/␮l)
Accuracy (%)
C0
30
150
500
7.5
37.5
125
1.5
7.5
25
261.8
87.4
89
20
150
300
5
37.5
75
1
7.5
15
1
7.5
15
1
7.5
15
1
7.5
15
1
7.5
15
1
7.5
15
1
7.5
15
1
7.5
15
2
15
30
2
15
30
2
15
30
2
15
30
119.7
95.5
97.8
116.1
97.7
97.9
116.3
94.2
97.8
115.5
92.2
93.3
120.0
93.3
93.9
113.9
89.5
96.0
108.3
91.8
97.6
115.4
102.6
100.6
110.1
104.7
100.9
109.8
103.6
106.6
108.7
107.5
105.7
119.1
109.2
105.8
112.3
102.4
102.1
107.8
106.0
104.0
C2
106.7
102.7
97.1
104.2
98.1
98.7
96.2
101
C3:0
C4:0
1.5
7.5
25
101
2-M-C3:0
3-M-C4:0
C5:0
1.5
7.5
25
1.5
7.5
25
1.5
7.5
25
1.5
7.5
25
82.9
97.2
96.4
84.3
94.3
94.2
84.1
95.8
94.1
90.8
100.8
102
C6:0
C8:0
1.5
7.5
25
1.5
7.5
25
3
15
50
3
90.3
105
107.9
86.3
99.3
98.1
87.2
95.8
96.5
89.3
91.7
90
C10:0
C12:0
C14:0
C16:0
C18:0
15
50
3
15
50
3
15
50
81.7
90.3
92.4
82.5
99.6
98.5
The displayed accuracy is the average of the assayed concentration
(corrected by endogenous levels of plasma and liver samples) in
percent of the actual spiked concentration. Concentrations in liver are
given per milligram of liver wet weight. w.w., wet weight.
acid oxidation associated with the metabolic syndrome
(5–7). Moreover, the metabolites were proposed to have
predictive quality for disease initiation and progression
(5). Various acylcarnitines occur as positional isomers and
many species often appear at very low concentrations. Even
though several LC-MS/MS methods have been described
for quantification of isomeric acylcarnitine species (9–12),
a comprehensive method that covers low-abundant spe-
cies and includes odd-numbered acylcarnitines is lacking.
Here we describe a sensitive and robust method for
quantification of individual isomeric acylcarnitine species,
including low-abundant amino acid-derived forms (see
Fig. 4). In addition, our method includes acylcarnitines
with odd-numbered acyl-chain lengths, which gained in-
creased attention recently as metabolite markers in dis-
ease states (5, 22, 23).
To evaluate LC-MS/MS method performance, we vali-
dated our method in plasma and liver samples as described
previously (26). Precisions showed CVs <15% for high-
abundant metabolites and CVs <20% for low-abundant
compounds. Accuracies displayed CVs of 10% with a
few exceptions. This is a major improvement over previ-
ously published methods, for which up to 30% varia-
tion was reported in plasma (9), or for which no measures
were reported at all (11). We observed that plasma sam-
ples were stable when stored for 1 h at room temperature
prior to preparation. Furthermore, most acylcarnitine
species in plasma and liver showed a high postpreparative
stability.
We applied ESI in positive mode for ionization. Colli-
sion-induced dissociation produced an intense product
An LC-MS/MS method to quantify acylcarnitine isomers
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TABLE 4. Intra- and inter-day precisions
Intra-day Liver (n = 5)
Mean SD
Inter-day Liver (n = 5)
Mean SD
Intra-day Plasma (n = 5)
Inter-day Plasma (n = 5)
Mean SD (pmol/␮l) CV (%)
Name
(pmol/mg w.w.)
CV (%)
(pmol/mg w.w.)
CV (%)
Mean SD (pmol/␮l)
CV (%)
C0
230.91 17.61
0.0098 0.001
1.62 0.099
3.89 0.21
0.17 0.006
60.01 5.23
0.68 0.056
12.73 1.31
0.14 0.013
0.0046 0.000
0.051 0.004
0.010 0.001
1.29 0.10
7.6
14.6
6.1
5.5
4.2
8.7
8.3
10.3
9.8
8.4
228.47 9.35
0.013 0.001
1.71 0.075
4.09 0.10
4.1
13.2
4.4
2.5
5.5
3.8
2.3
3.8
2.2
11.5
4.0
13.2
7.0
4.1
7.3
13.8
3.8
5.0
4.6
12.2
4.7
2.6
4.6
10.0
2.3
8.1
4.2
9.3
6.8
4.1
3.2
—
5.7
9.2
34.99 3.29
—
9.4
—
12.7
11.7
14.2
8.0
10.6
9.7
10.4
11.0
12.3
—
33.34 2.01
—
6.1
—
15.4
7.8
8.3
4.6
14.3
9.7
14.5
11.4
15.2
—
10.3
9.3
12.9
—
12.4
11.3
11.0
14.2
11.0
9.3
10.3
16.8
11.0
12.1
—
C3-OH
C4-OH a
C4-OH b
2-M-C3-OH
C2
0.051 0.01
0.10 0.01
0.062 0.00
16.73 1.34
0.067 0.00
0.52 0.05
0.005 0.00
0.003 0.00
0.005 0.00
—
0.16 0.01
0.45 0.04
0.005 0.00
—
0.054 0.00
0.070 0.00
0.011 0.00
0.001 0.00
0.039 0.00
0.029 0.00
0.068 0.00
0.010 0.00
0.032 0.00
0.017 0.00
—
0.043 0.00
0.001 0.00
0.011 0.00
0.009 0.00
0.004 0.00
—
0.049 0.01
0.098 0.01
0.059 0.00
15.34 0.69
0.067 0.00
0.51 0.04
0.005 0.00
0.003 0.00
0.005 0.00
—
0.15 0.01
0.43 0.03
0.005 0.00
—
0.054 0.00
0.070 0.00
0.011 0.00
0.001 0.00
0.038 0.00
0.029 0.00
0.066 0.00
0.011 0.00
0.030 0.00
0.020 0.00
—
0.039 0.00
0.001 0.00
0.010 0.00
0.008 0.00
0.007 0.00
—
0.17 0.009
61.26 2.34
0.67 0.015
12.69 0.48
0.14 0.003
0.0046 0.000
0.051 0.002
0.0099 0.001
1.35 0.094
5.40 0.22
0.011 0.000
0.0049 0.000
0.70 0.026
0.18 0.008
1.56 0.072
0.025 0.002
4.78 0.23
1.49 0.039
1.31 0.059
0.11 0.010
5.84 0.13
0.92 0.074
4.81 0.20
0.097 0.009
0.20 0.013
2.53 0.10
0.20 0.006
—
0.038 0.002
0.092 0.008
0.0086 0.000
0.0060 0.001
0.044 0.005
0.082 0.002
0.033 0.004
0.0039 0.000
0.0039 0.000
0.087 0.003
0.21 0.009
0.049 0.002
0.0098 0.001
0.13 0.013
0.57 0.021
0.030 0.001
0.011 0.001
1.50 0.049
1.35 0.050
2.74 0.045
0.018 0.000
0.025 0.001
0.0045 0.001
0.93 0.038
C5-OH
C3:0
C6-OH a
C4:1
C6-OH b
2-M-C3:1
2-M-C3:0
C4:0
2-M-C4:1
3-M-C4:1
2-M-C4:0
3-M-C4:0
C5:0
C4:1-DC
C3-DC
C4-DC
C6:0
C3-DC-M
C5-DC
C5:1-DC
C6-DC
C5-M-DC
C7:0
9.3
11.9
8.0
9.5
8.9
8.9
—
5.34 0.45
8.5
0.011 0.001
0.0053 0.000
0.72 0.060
0.18 0.018
1.56 0.17
0.024 0.002
4.61 0.44
1.51 0.16
1.33 0.13
0.11 0.010
5.87 0.53
0.91 0.16
17.8
13.8
8.4
10.1
10.8
11.4
9.5
10.6
10.0
9.6
12.3
9.7
11.4
12.7
11.1
8.8
10.9
18.6
11.7
13.2
—
10.7
19.9
9.1
9.0
11.8
—
13.6
—
—
9.0
17.1
10.9
15.3
14.0
10.3
8.9
4.92 0.53
0.095 0.014
0.19 0.026
2.59 0.27
0.19 0.017
—
8.1
17.0
10.8
12.5
19.5
—
14.9
—
C7-DC
C8:0
C10:1
C9:0
C10:0
Iso-C11:0
C11:0
C14:2
—
0.038 0.003
0.10 0.010
0.0094 0.000
0.0055 0.000
0.047 0.002
0.090 0.011
0.031 0.013
0.0035 0.000
0.0035 0.000
0.089 0.009
0.22 0.021
0.050 0.005
0.0098 0.001
0.11 0.022
0.54 0.053
0.011 0.001
0.012 0.000
1.45 0.18
10.3
10.9
17.7
18.6
5.1
13.2
44.6
20.0
18.4
10.3
9.5
10.3
13.4
19.5
10.0
15.2
6.5
0.010 0.00
—
0.009 0.00
—
3.1
12.7
12.5
3.1
12.5
14.2
14.2
4.4
4.3
4.7
11.1
10.6
3.8
3.8
9.6
3.3
3.7
1.7
4.4
6.9
24.1
4.1
—
—
—
0.001 0.00
0.015 0.00
—
0.000 2.37
0.001 0.00
0.011 0.00
0.048 0.00
0.003 0.00
0.002 0.00
—
0.049 0.00
0.001 0.00
0.002 0.00
0.051 0.01
0.18 0.03
0.11 0.02
0.001 0.00
0.001 0.00
—
10.9
19.9
—
0.001 0.00
0.013 0.00
—
0.000 3.54
0.001 0.00
0.010 0.00
0.044 0.00
0.010 0.00
0.002 0.00
—
0.045 0.01
0.000 0.00
0.002 0.00
0.051 0.01
0.17 0.03
0.11 0.03
0.001 0.00
0.001 0.00
—
19.9
19.8
—
12.6
16.8
25.3
19.6
24.7
23.7
—
31.1
20.0
13.5
38.7
18.5
36.9
17.4
14.0
—
C12:0
C16:1-OH
Iso-C13:0
C13:0
C16:2
C14:0
C16-OH a
C16-OH b
C18:1-OH
C16:1
Iso-C15:0
C15:0
C18:2
C16:0
C18:1
Iso-C17:0
C17:0
C12-DC
C18:0
7.9
18.8
18.4
16.5
19.0
16.3
—
18.9
18.7
15.2
20.9
19.5
22.6
16.3
16.1
—
12.6
8.3
9.4
1.40 0.12
2.67 0.25
0.019 0.001
0.029 0.004
0.0048 0.000
0.91 0.081
9.8
14.2
18.2
9.0
0.028 0.00
17.8
0.027 0.00
19.2
Intra- and inter-day precisions were assessed by calculating average concentrations and CVs from five individual plasma and liver samples.
Concentrations in liver are given per milligram of liver wet weight. w.w., wet weight.
We applied our method to plasma and liver samples of
STZ-treated insulin-deficient mice. STZ is selectively taken
up into the pancreas and causes ␤-cell toxicity via alkylation,
resulting in ␤-cell necrosis (20). STZ-treated mice showed
very low circulating insulin levels paralleled by a strong in-
crease in blood glucose and a dramatic decrease in body
weight (see supplementary Table 5).
We found that, particularly, the branched-chain acylcarni-
tines, as intermediates of branched-chain amino acid degra-
dation, were increased in plasma and liver, and among them,
moststrongly,propionyl-carnitine(C3:0-CN),2-methylbutyryl-
carnitine (2-M-C4:0-CN), and isovaleryl-carnitine (3-M-C4:0-
CN), as well as 2-methylcrotonyl-carnitine (2-M-C4:1-CN)
and 3-methylcrotonyl-carnitine (3-M-C4:1-CN). Interestingly,
2036
Journal of Lipid Research Volume 56, 2015

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Fig. 3. Levels of acylcarnitine species in plasma and liver tissue samples obtained from STZ-treated animals and controls determined with
our LC-MS/MS method. A, D: High-abundant branched-chain amino acid-derived species. B, E: Low-abundant branched-chain amino acid
derived species. C, F: Odd-numbered species. Concentrations in liver are given per milligram of liver wet weight. White and black bars
represent metabolite concentrations in controls and diabetic animals, respectively. *P < 0.05, **P < 0.01, and ***P < 0.005.
we also detected elevated levels of the 5-carbon straight-
chain valeryl-carnitine (C5:0-CN), for which the metabolic
origin is obscure. Increases in plasma levels of 3-carbon-
chain and 5-carbon-chain acylcarnitines derived from
breakdown of branched-chain amino acids, as deter-
mined by a direct infusion MS/MS approach, were re-
cently reported as important predictors for type 2
diabetes (5). Furthermore, while concentrations of 3-hy-
droxybutyryl-carnitine (C4-OH-CN) were strongly in-
creased (by ف40%), in diabetic mice, probably as a result
of increased ketone body production (27), levels of 3-hy-
droxyisobutyryl-carnitine (2-M-C3-OH-CN), an interme-
diate in valine breakdown, displayed a strong decrease
(by ف50%) in both plasma and liver. For a correct inter-
pretation of changes in these different pathways, it is
thus necessary to include LC separation prior to MS for
individual quantification of these metabolites. Similarly,
while using direct infusion MS/MS, Mihalik et al. (6) re-
ported C4-dicarboxyl-carnitine as a marker for glucoli-
potoxicity in type 2 diabetes, but did not discriminate
between succinyl-carnitine (C4-DC-CN) and methylmal-
onyl-carnitine (C3-M-DC-CN), which are interconverted
via methylmalonyl-CoA mutase. With our method, we
found that only levels of methylmalonyl-carnitine (C3-M-
DC-CN) were significantly increased in diabetic mice,
while alterations in succinyl-carnitine (C4-DC-CN) con-
centrations remained insignificant. Finally, we found ele-
vated levels of several odd-numbered acylcarnitines in
diabetic animals, most significantly the short-chain spe-
cies, propionyl-carnitine (C3-CN), valeryl-carnitine (C5-CN),
and heptanoyl-carnitine (C7-CN) in liver. Interestingly, odd-
numbered fatty acids were recently proposed as potential
markers of the metabolic syndrome (22). The origin and
cause for these alterations in disease states is not known.
In summary, we have developed a sensitive and robust
method which allows quantification of a large number of
acylcarnitine species, including isomeric forms derived
from a variety of metabolic pathways in amino acid and
fatty acid oxidation. Compared with existing methods, we
expanded the palette of analytes with novel acylcarnitine
species, including low-abundant odd-numbered forms.
Our LC-MS/MS method was validated in plasma and liver
samples from mice and was applied to samples from STZ-
treated type I diabetic mice. These mice display increased
concentrations of BCAA-derived and odd-numbered acyl-
carnitine species, which were recently proposed as poten-
tial biomarkers for the metabolic syndrome.
The authors thank Mena Eidens for excellent participation in
conduction of mouse experiments and sample collection.
An LC-MS/MS method to quantify acylcarnitine isomers
2037

Supplemental Material can be found at:
http://www.jlr.org/content/suppl/2015/08/03/jlr.D061721.DC1
.html
Fig. 4. Scheme displaying amino acid breakdown pathways and acylcarnitine species that can be formed
from acyl-CoA intermediates.
separation of 48 acylcarnitines in dried blood spots and plasma
useful as a second-tier test for expanded newborn screening. Anal.
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