Determination of idebenone in plasma by HPLC with post-column fluorescence derivatization using 2-cyanoacetamide

Article abstract of DOI:10.1248/cpb.60.598

Idebenone is a benzoquinone analog that is used in the treatment of several neurological disorders including Friedreich's ataxia. It was found that the reaction of idebenone with 2-cyanoacetamide under alkaline conditions generates fluorescent products, a

Full text of DOI:10.1248/cpb.60.598

598
Regular Article
Chem. Pharm. Bull. 60(5) 598–602 (2012)
Vol. 60, No. 5
Determination of Idebenone in Plasma by HPLC with Post-Column
Fluorescence Derivatization Using 2-Cyanoacetamide
,a
Yukio Nohara,* Junko Suzuki,b Yoji Yamazaki,a and Hiroaki Kuboa
b
a Faculty of Pharmacy, Iwaki Meisei University; Iwaki, Fukushima 970–8551, Japan: and School of Pharmacy,
Kitasato University; Minato-ku, Tokyo 108–8641, Japan.
Received November 18, 2011; accepted February 8, 2012; published online February 9, 2012
Idebenone is a benzoquinone analog that is used in the treatment of several neurological disorders in-
’
cluding Friedreich s ataxia. It was found that the reaction of idebenone with 2-cyanoacetamide under alkaline
’
conditions generates fluorescent products, and the reaction was considered to proceed via Craven s reaction.
The reaction mixture from idebenone gave fluorescence with excitation and emission maximum wavelengths
at 358nm and 409nm, respectively. It was adopted for HPLC with post-column fluorescence derivatization
of idebenone. Idebenone in the plasma showed a linear response in the range of 0.5–32ng (25–1600ng/mL),
and the quantitation limit (S/N=10) was 12.5ng/mL. The detection limit (S/N=3) of the standard solution of
idebenone was 0.1ng. The HPLC system was applied to the human blood plasma obtained by finger prick.
The plasma sample obtained by finger prick gave a similar chromatogram to that of venous blood obtained
by venipuncture.
Key words idebenone; finger prick; 2-cyanoacetamide; fluorescence derivatization; HPLC
Idebenone is a synthetic analog of ubiquinone,1) which is containing potassium fluoride.16) It was reported that cortisol
an essential component of the mitochondrial respiratory chain and cortisone in umbilical cord blood were prepared using
responsible for oxidative phosphorylation, and may prevent acetonitrile for deproteinization with anhydrous sodium car-
oxidative damage in the human body as an antioxidant.2,3) bonate and sodium chloride for separation of water layer and
Idebenone also has potent antioxidant activity and facilitates acetonitrile layer as a sample for chemiluminescence HPLC.17)
the flux of electrons along the mitochondrial electron transport The conventional pretreatment methods of plasma or serum
chain, increasing the production of ATP, and has already been for HPLC analysis of idebenone are complicated.6–9) These
clinically investigated for the treatment of several neurologi- methods need steps of extraction by organic solvent after the
’
’
cal disorders, which include Friedreich s ataxia and Leber s hydrolysis of conjugate in plasma or serum sample by hydro-
hereditary optic neuropathy.4,5)
chloric acid, and evaporation of organic solvent layer; finally,
The pharmacokinetic properties of idebenone have been in- the residue is dissolved in solvent as sample solution. In this
vestigated by HPLC. Several procedures have been developed study, after the hydrolysis of plasma sample by hydrochloric
to analyze idebenone in biological samples by HPLC coupled acid, the sample pretreatment by extraction of idebenone in
with UV, electrochemical, or mass spectrometry detection.6–9) plasma using acetonitrile with anhydrous sodium carbonate
HPLC methods employing mass spectrometry and electro- was effective for excellent separation of idebenone from other
chemical detectors have higher selectivity and sensitivity fluorescent substances in blood; moreover, this method had a
than those with a UV detector; however, these methods are simple and rapid form of pretreatment.
not available in many laboratories because of a lack of spe-
A sample of blood obtained by finger prick is useful for
cialized equipment. HPLC coupled with a fluorophotometric rapid and easy assay. Because self-collection of blood by
detector has been widely used in the determination of various finger prick with an automatic lancet is not very invasive
substances in biological samples because of its high sensitiv- and easy to perform, it is useful for self-monitoring, such
ity and selectivity. However, idebenone needs fluorescence as for blood glucose level, hemoglobin A1c and lactate.18‒20)
derivatization for the development of fluorimetric detection Moreover, dried blood spots obtained by finger prick are used
because it is not a fluorescent substance itself. 2-Cyanoacet- for the therapeutic drug monitoring (TDM) of antibiotics,
amide has been used as a post-column derivatization reagent anti-epileptics, analgesics, benzodiazepines, and others.21–24)
of fluorimetric HPLC for catecholamines, 3,4-dihydroxyphe- However, analysis of serum and plasma idebenone derived
nylalanine, unsaturated disaccharides, and benzoquinones.10–15) from venous blood by HPLC is conventionally used, and the
In the present study, 2-cyanoacetamide was tested for the process of sampling blood by venipuncture is invasive and re-
fluorophotometric reagent of idebenone because idebenone is a quires blood-drawing techniques using syringes. In this paper,
benzoquinone compound. It was found that idebenone reacted the application of this method to the detection of idebenone in
with 2-cyanoacetamide under alkaline conditions with heat- plasma obtained by finger prick is described.
ing to form fluorescent products. This reaction was applied
to HPLC with post-column fluorescence derivatization of
Experimental
idebenone.
Materials Water was purified using a MILLI-Q Labo
The salting out effect is useful as a simple and rapid meth- from Nihon Millipore Kogyo (Yonezawa, Japan). 2-Cyano-
od for the pretreatment of biological samples. The addition of acetamide was purchased from Tokyo Kasei Kogyo (Tokyo,
a salt to a mixture of water and aliphatic alcohol occurs in Japan). Idebenone, sodium perchlorate, hydrochloric acid, ace-
two phase equilibrium of water and alcohol layer, for instance, tonitrile, methanol, and HPLC-grade ethanol were from Wako
the combination of 1-propanol (or 2-propanol) and water Pure Chemical Industries (Osaka, Japan). Sodium hydroxide
*To whom correspondence should be addressed. e-mail: ynohara@iwakimu.ac.jp
© 2012 The Pharmaceutical Society of Japan

May 2012
599
and anhydrous sodium carbonate were from Kanto Chemical collected in a tube with 1mg of ethylenediaminetetraacetic
(Tokyo, Japan). All other chemicals were of analytical grade. acid (EDTA). The blood was then centrifuged at 3000rpm
Fluorophotometric Determination Five hundred micro- for 15min at 4 C within 1–3h, and the plasma was stored at
°
°
liters of 6mmol/L 2-cyanoacetamide aqueous solution con- −80 C until analysis.
taining 6mmol/L sodium perchlorate, 20µL of 2mmol/L
A 50µL sample of plasma was vortex-mixed with 50µL of
idebenone in ethanol, and 605µL of ethanol were mixed, and 6mmol/L hydrochloric acid for 20s. The mixture was heated
°
then 125µL of 160mmol/L sodium hydroxide aqueous solu- at 60 C for 20min, then cooled on ice. One hundred and fifty
tion was added to the mixture. The mixture was heated at microliters of acetonitrile was added to the mixture, and then
°
60 C for 5min, then after cooling on ice, the excitation and the mixture was vortex-mixed for 10s. Moreover, 50mg of an-
emission spectra were measured using a spectrofluorometer hydrous sodium carbonate was added to the mixture, and then
(RF-5000, Shimadzu, Kyoto, Japan).
the mixture was vortex-mixed for 20s. Finally, the mixture
Apparatus and Chromatographic Conditions The liquid was centrifuged at 10000rpm for 60s, and the upper layer of
chromatograph consisted of two isocratic pumps (PU-2080 20µL was injected onto an HPLC directly.
Plus, JASCO, Tokyo, Japan), manual sample injector with 20-
Preparation of Reduced Form of Idebenone The re-
µL loop (7725i, Rheodyne, U.S.A.), column oven (655-A52, duced form of idebenone standard solution was prepared from
Hitachi, Tokyo, Japan), reaction oven (S-3850, Soma Optics, reduction of idebenone by sodium borohydride such as reduc-
Tokyo, Japan), and integrator (807-IT, JASCO, Tokyo, Japan). tion of ubiquinone.25) First, 890µL of ethanol and 100µL of
Detection was performed with a fluorescence detector (FP- idebenone in ethanol were mixed in a tube, and then 10µL
2020 Plus, JASCO, Tokyo, Japan).
of 0.25% sodium borohydride aqueous solution was added to
The eluent was a mixture of ethanol aqueous solu- the mixture. The mixture was left to stand for 15min at room
tion (65v/v%) containing 3mmol/L 2-cyanoacetamide and temperature, and the reduced form of idebenone standard so-
3mmol/L sodium perchlorate. The eluent was delivered from lution was used immediately. The reduction rate of idebenone
a pump at a flow rate of 0.8mL/min. A 20µL sample was in- to reduced form of idebenone, which has a maximum absorp-
jected via the injector. The idebenone from other fluorescent tion wavelength at 275nm, was determined by HPLC.
substances in plasma was separated in the octadecylsilane
(ODS) column (Inertsil ODS-3, 150×4.6mm i.d., particle size
Results and Discussion
Fluorophotometric Detection It is known that Craven s
°
’
5µm) at 25 C. The eluate was mixed with 80mmol/L sodium
hydroxide in ethanol aqueous solution (65v/v%) as alkaline reaction, which is a specific color reaction for quinone, pro-
solution delivered from another pump at a flow rate of 0.2mL/ duces transitory blue-colored products by the displacement
min. The mixture was reacted in a coil (5m×0.5mm i.d.) of methoxyl substituents, such as the structure of ubiquinone,
°
then placed in the oven at 60 C. After cooling in a coil (3m with active methylene compounds, such as ethyl cyanoacetate,
× 0.5mm i.d.), the fluorescence intensity was monitored with under alkaline conditions.26–29) Moreover, Craven s reaction
’
the fluorescence detector (excitation at 358nm, emission at gives fluorescent products at a later stage of the reaction. It
409nm, slit: 18nm, response: slow, gain: 10).
was reported that ubiquinone incubated at room temperature
Pretreatment of Plasma Samples Collected by Fin- for 12–24h in ethyl cyanoacetate with methanolic potassium
ger Prick Idebenone was administered orally as 150mg hydroxide solution resulted in fluorescent products with ex-
(30mg×5) of tablets (Cerestabon, Takeda, Japan) by two male citation at 430nm and emission at 530nm.28) It was shown
healthy volunteers aged 68 and 41. Fasting blood samples that ubiquinone produces fluorescent products under alkaline
were obtained by finger prick before the administration of conditions when reacted with 2-cyanoacetamide, which is also
idebenone and 1h thereafter. The blood samples of the healthy an active methylene compound with a cyano group, instead of
volunteers were included after informed consent had been ob- ethyl cyanoacetate.15) This reaction was considered to proceed
’
tained. Forty microliters of blood was collected in a heparin- via Craven s reaction. Figure 1 shows excitation and emission
treated hematocrit capillary tube (VC-H075H, TERUMO, spectra of idebenone after the reaction with 2-cyanoacet-
Tokyo, Japan) by finger prick using a lancing device with a amide containing ethanol aqueous solution (50v/v%) under
lancet corresponding to 23 gauge (FINETOUCH, TERUMO, alkaline conditions. The excitation and emission maximum
Tokyo, Japan). The blood was then centrifuged at 10,000rpm wavelengths from the mixture after the reaction were 358nm
for 60s at room temperature within 1–3h, and 15µL of the and 409nm, respectively. The fluorescence was shown in the
°
plasma was stored at −80 C until analysis.
presence of idebenone. This finding was considered to show
’
A 15µL sample of plasma was vortex-mixed with 15µL of that the reaction proceeds through Craven s reaction, such
6mmol/L hydrochloric acid for 20s. The mixture was heated as reaction of ubiquinone with 2-cyanoacetamide. Figure 2
°
at 60 C for 20min, then cooled on ice. Forty-five microliters shows a scheme of the production of fluorescent products by
of acetonitrile was added to the mixture, and then the mix- the reaction of idebenone and reduced form of idebenone with
ture was vortex-mixed for 10s. Moreover, 15mg of anhydrous 2-cyanoacetamide under alkaline conditions. It was reported
sodium carbonate was added to the mixture, and then the that two fluorescent compounds, which were considered to be
mixture was vortex-mixed for 20s. Finally, the mixture was 3-cyano-2-pyridone and 3-cyano-2-pyrrolidone derivatives,
centrifuged at 10000rpm for 60s, and the upper layer of 20µL were isolated from the reaction mixture of glucose and 2-cya-
was injected onto an HPLC directly.
noacetamide.30) This finding indicated that the fluorescent
Pretreatment of Plasma Samples Taken from the An- products by the reaction of idebenone with 2-cyanoacetamide
tecubital Vein Fasting blood samples were taken from the may be pyridone or pyrrolidone derivatives.
antecubital vein using a syringe before the administration
It was reported that the effect of solvents on the fluores-
of idebenone and 1h thereafter. One milliliter of blood was cence emission spectra of N,N-dimethylamino-4-(2-cyano-

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Vol. 60, No. 5
2-acetamidovinyl)-benzene, which was synthesized from and 2-propanol showed the same spectrum as ethanol (Fig.
p-N,N-dimethylamino-benzaldehyde and 2-cyanoacetamide, 1). The emission spectra of the reaction mixture containing
and the fluorescence maxima were red-shifted with increasing acetonitrile showed a shoulder around 500nm. Moreover, the
solvent polarity.31) In this study, the fluorescence spectra and emission spectra of the reaction mixture containing methanol
intensity from the reaction mixture of idebenone with 2-cya- showed a broad peak at about 410nm. The addition of water or
noacetamide were also affected by organic solvents and water acetone to the reaction mixture did not produce fluorescence.
(their solvent volume was 50v/v%). The emission spectra were The eluent composition of ethanol aqueous solution was used
measured at an excitation wavelength of 358nm. 1-Propanol for HPLC because it showed the highest fluorescence intensity
at 409nm for the solvents, and the excellent separation of ide-
benone from other fluorescent substances in blood.
Fluorimetric HPLC for Idebenone The reaction of 2-cy-
anoacetamide with idebenone under alkaline conditions was
available for HPLC with post-column fluorescence derivatiza-
tion. The chromatographic conditions were optimized using
a standard solution of idebenone. The optimal concentration
of reagents for the fluorescence derivatization was investi-
gated over the ranges of 0.5–4mmol/L for 2-cyanoacetamide,
and 20–100mmol/L for sodium hydroxide. The maximum
fluorescence intensities for the fluorescence of idebenone were
obtained at concentrations of 3mmol/L 2-cyanoacetamide
and 80mmol/L sodium hydroxide. The reaction temperature
°
was investigated over the range of 50–75 C. The baseline was
°
unstable over a temperature of 65 C and the maximum peak
°
area was observed at 60 C. In addition, 3mmol/L sodium
perchlorate as an anionic ion pair reagent in the eluent was
effective for excellent separation of idebenone from other
fluorescent substances in blood.7,32) On the other hand, 2-cya-
noacetamide was soluble and stable in the eluent; therefore,
Fig. 1. The Fluorescence Excitation (Ex) and Emission (Em) Spectra of
0.03mmol/L Idebenone Allowed to React with 2.4mmol/L 2-Cyanoacet-
amide, 16mmol/L Sodium Hydroxide, and 2.4mmol/L Sodium Perchlo-
°
rate in Ethanol (50v/v%) at 60 C for 5min
Fig. 2. Possible Mechanism of Fluorescence Derivatization by the Reaction of Idebenone and Reduced Form of Idebenone with 2-Cyanoacetamide
under Alkaline Conditions

May 2012
601
Fig. 3. Chromatograms of Standard Solution of Idebenone (2.0ng/Injection) (A), Standard Solution of Idebenone (3.6ng/Injection) Plus Reduced
Form of Idebenone (1.4ng/Injection) (B), Plasma Plus Standard Solution of Idebenone (2.0ng/Injection) (C), Plasma from the Antecubital Vein of a
Healthy Volunteer before the Administration of Idebenone (D) and after 1h (E)
this post-column derivatization HPLC system was simplified at concentrations of 200ng/mL and 800ng/mL under the same
because only two pumps were used to deliver the eluent con- conditions were 98.1% and 98.3%, respectively.
taining 2-cyanoacetamide and the alkaline solution.
Figures 3D and E show chromatograms of plasma from
Figure 3A shows a chromatogram of the standard solution the antecubital vein of a healthy volunteer before the oral
of idebenone. The detection limit (S/N=3) of idebenone was administration (150mg) of idebenone and at 1h, respectively.
0.1ng. Figure 3B shows a chromatogram of the standard solu- Administered idebenone in human is metabolized by oxidative
tion of idebenone plus the reduced form of idebenone. It was shortening of the 10-hydroxydecyl side chain, and reduction of
found that the reduced form of idebenone reacted with 2-cya- the quinone ring and subsequent conjugation yielding the sul-
noacetamide under alkaline conditions the same as idebenone. fates and glucuronides of the hydroquinone derivatives of the
The reduced form of idebenone in the reaction coil of HPLC parent compound and the oxidized metabolites.34,35) The total
may react with 2-cyanoacetamide via idebenone such as ubi- idebenone, which is the sum of parent idebenone and decon-
quinol because ubiquinol, which is a reduced form of ubiqui- jugated idebenone by the acid treatment, in human blood was
none, was oxidized under alkaline conditions with heating.33)
determined in this study. Idebenone was not detected, and an
Figure 3C shows a chromatogram of plasma plus standard unknown peak of other fluorescent substances was detected,
solution of idebenone. Idebenone showed a linear response in as shown in Fig. 3D. In contrast, total idebenone was detected
the range of 0.5–32ng (25–1600ng/mL) and the correlation co- and separated from other fluorescent substances, which was
efficient of the curve was more than 0.9980. The quantitation achieved within 10min as shown in Fig. 3E. The mean plasma
limit (S/N=10) of idebenone was 12.5ng/mL. The within-run level from two healthy male volunteers orally administered
(n=6) coefficients of variation at concentrations of 200ng/mL idebenone (150mg) at 1h was 422.8ng/mL.
and 800ng/mL were 1.70% and 2.72%, and those from day to
Figure 4 shows a chromatogram of plasma obtained by
day (n=5) were 3.79% and 3.95%, respectively. The recoveries finger prick at 1h from a healthy volunteer administered

602
Vol. 60, No. 5
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2-Cyanoacetamide was shown to be a useful reagent for
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