Journal of Pharmaceutical and Biomedical Analysis 88 (2014) 331–336
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis
journal homepage: www.elsevier.com/locate/jpba
Pharmacokinetics study of hemin in rats by applying 58 Fe-extrinsically
labeling techniques in combination with ICP-MS method
Di Zhao a,d , Yongjie Zhang a , Yue Wang b , Chunxiang Xu c , Can Dong c , Cuiyun Li a ,
Shuangxia Ren a , Wei Zhang a , Yang Lu a , Yue Dai d,∗ , Xijing Chen a,∗∗
a
Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009, China
State Key Laboratory of Natural Medicines, School of Basic Science, China Pharmaceutical University, Nanjing 210009, China
Jiangsu Product Quality Test & Inspect Institute, Nanjing 210007, China
d
Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing 210009, China
b
c
a r t i c l e
i n f o
Article history:
Received 9 May 2013
Received in revised form 27 August 2013
Accepted 28 August 2013
Available online 9 September 2013
Keywords:
58
Fe
Hemin
ICP-MS
Pharmacokinetics
Iron absorption
Extrinsically labeling
a b s t r a c t
Iron is a challenging element due to its high background in various matrixes including blood, tissues
even in the air and it is urgent to develop a method for the accurate determination of iron in bio-samples.
After optimization of mass spectrometric conditions using collision cell technology and compensating for
interference using a mathematical correction equation, an inductively coupled plasma mass spectrometry (ICP-MS) method for the quantitative determination of 58 Fe originating from hemin extrinsically
labeled avoiding endogenous interference was developed. After a single step of dilution, analysis of each
sample was completed within 1.5 min. The assay was linear in the concentration range from 0.005 to
1.0 ␮g/ml. The precisions and accuracies determined within three consecutive days were in acceptable
limits and there was no significant matrix effect. The optimized method was successfully applied to a
pharmacokinetic study of 58 Fe originating from hemin extrinsically labeled and iron absorption measured in rats was 1.07%. Those indicated that extrinsically label techniques in combination with ICP-MS
will become a new tool for the analysis of iron preparations and other endogenous substances.
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
Iron is one of the important essential trace elements in the
human body. Iron deficiency is currently the most common
micronutrient deficiency worldwide affecting more than 40% of
women at child-bearing age in developing countries [1]. To reduce
the prevalence and severity of iron deficiency, many countries such
as Mexico [2], Sri Lankan [3], and Peruvian [4] require iron supplements into their daily diet. There are various iron preparations
on the market and they can be either heme-iron or nonhemeiron and heme-iron absorption was considered to be greater than
nonheme-iron absorption [5]. The iron in heme is in its oxidized
state, while it is in reduced state in hemin, iron (III) protoporphyrin
chloride (IX) (chemical structure shown in Fig. 1) [6]. That means
the use of hemin in absorption study of iron instead of heme is very
∗ Corresponding author at: Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing 210009, Jiangsu, China.
Tel.: +86 25 83271400; fax: +86 25 85301528.
∗∗ Corresponding author at: Center of Drug Metabolism and Pharmacokinetics,
China Pharmaceutical University, Nanjing 210009, Jiangsu, China.
Tel.: +86 25 83271286; fax: +86 25 83271335.
E-mail addresses: [email protected] (Y. Dai), [email protected]
(X. Chen).
0731-7085/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jpba.2013.08.048
meaningful considering the instability of heme molecule, and this
view is supported in Villarroel and Jahn’s in vitro study [6,7]. One of
the first papers to describe intrinsic labeling using stable isotope for
iron absorption measurement. However, it is difficult to evaluate
the absorption of iron from hemin in vivo because of its high background in blood. Thus the determination of iron in bio-samples is
considered to be of great importance.
Presently the most widely used method is probably based on the
quantitation of elemental iron by atomic absorption spectroscopy
[8–10]. However, one of the major limitations of these methods
is the lack of adequate sensitivity required for pharmacokinetic
studies. For example, the limit of quantitation for iron was only
1.0 ␮g/ml and the analytical range was just from 1.0 to 4.0 ␮g/ml
in Shang’s method [8]. Due to the poor sensitivity, it is very difficult
to extend these methods to bio-sample analysis. Also, these methods were developed for the quantitative determination of iron in
food or pharmaceutical preparations while iron is a challenging element due to the substantially high background in various matrixes
including blood, tissues even in the air and water and it is very
difficult to distinguish between endogenous and exogenous iron.
Therefore, a method using labeled iron as a tracer is required for
the accurate determination of iron in bio-samples.
Radioisotopes of iron including 55 Fe and 59 Fe were usually
used as tracers in the research of uptake and metabolism of iron
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D. Zhao et al. / Journal of Pharmaceutical and Biomedical Analysis 88 (2014) 331–336
2. Materials and methods
2.1. Chemicals and reagents
Stable isotope, iron-58, as metal powder (>99.86%, enrichment),
was obtained from ISOFLEX USA (San Francisco, CA, USA). Hemin
extrinsically labeled with 58 Fe was supplied by State Key Laboratory
of Natural Medicines, School of Basic Science, China pharmaceutical university. Germanium Plasma Emission Standard (>99.999%,
purity) was supplied by Shanghai ANPEL Scientific Instrument
Co., Ltd. (Shanghai, China). Triton X-100 of chemical purity and
Nitric acid of MOS grade were from Sinopharm Chemical Reagent
Company (Shanghai, China). N-butyl alcohol of analytical grade
was purchased from Nanjing Chemical Reagent Co., Ltd. (Nanjing,
China). Deionized water was purified using a Milli-Q system (Millipore, Milford, MA, USA). Standard stock solution of 58 Fe was
prepared in nitric acid (2%, v/v) at 2 mg/ml and germanium (Ge)
was in a solution (nitric acid 2%, v/v) at 1 mg/ml, then were serially
diluted to working solution with nitric acid (2%, v/v). All the stock
and working solutions were stored at 4 ◦ C.
2.2. Apparatus
Fig. 1. The chemical structure of hemin. 夽: labeled by 58 Fe.
[11,12]. In Swati’s method, 59 Fe was successfully used to evaluate the effects of dietary factors on iron uptake from ferritin in
Caco-2 cells [13]. However, due to the risk associated with exposure to radiation, stable isotopes may be a better choice. Iron
has 4 naturally occurring isotopes, including 54 Fe (5.85%), 56 Fe
(91.75%), 57 Fe (2.12%) and 58 Fe (0.28%). The existence of polyatomic ion interferences particularly for 54 Fe, 56 Fe [14] and the
lowest abundance for 58 Fe in blood make it a good opportunity for
pharmacokinetic research [15]. While to our knowledge, no successful attempts to extrinsically label hemin with 58 Fe have been
published.
Since 1983, when inductively coupled plasma mass spectrometry (ICP-MS) became commercially available firstly [16], it has
been demonstrated to be a valuable tool for the determination of
drugs containing metals, due to its excellent sensitivity and selectivity [17]. What’s more, compared to negative thermal ionization
mass spectrometry (NTIMS), there are many advantages for ICP-MS,
such as multi-element capabilities, much easier sample preparation and the speed of isotopic analysis [18,19]. Recently a large
number of papers reporting the use of ICP-MS for iron isotope
ratio determinations have been published [20–22]. However, in
Whittaker’s method, the sample analysis was too time consuming
and the isotope ratio, not the isotope concentration was treated
as the information for absorption study. More importantly, this
method has not been validated systematically [14,23]. In fact, the
similar phenomenon was existed in Ronny Schoenberg’ research,
though extremely high precision has been gotten (95%CI of 0.05 per
mille). The method was mainly used to measure stable Fe isotope
ratio in natural samples not pharmacokinetic study in bio-samples
and there was no need to distinguish between endogenous and
exogenous iron [12]. Therefore, it is critical to develop a simple, sensitive analytical method for the determination of exogenous iron in
plasma in order to get more accurate information on the bioavailability and comprehensive understanding of the pharmacokinetic
behaviors of iron supplements.
The objectives of this study were firstly to develop and
validate a sensitive, specific and reliable ICP-MS method for
the determination of 58 Fe originating from hemin extrinsically
labeled in plasma and secondly to apply this method in a pharmacokinetic study of hemin and measure iron absorption in
rats.
All analytical experiments were carried out on an ICP-MS
(XSERIES 2, Thermo Scientific Waltham, MA, USA), which was
operated with the Plasma Screen Plus sensitivity enhancement
option fitted, Xt interface cones and with peltier cooling of the
spray chamber. A standard quartz nebulizer was used, together
with a standard quartz impact bead spray chamber, standard single piece, and 1.5 mm i.d. injector quartz torch. The instrument
was operated using collision cell technology (CCT) mode (using
7% (v/v) H2 in He as the collision gas). Plasma Lab software
was applied for instrument control, data acquisition and analysis.
2.3. Determination of 58 Fe by ICP-MS
The instrumental and operating condition were optimized
with the multi-element tune solution. Typically, this solution
gave readings of 115 In: >4 × 104 c/s; 59 Co: >1.5 × 104 c/s; 238 U:
>8.0 × 105 c/s. Performance was checked daily. Ge (50 ng/ml)
was added online using of three-way valve as internal standard.
The operating parameters of ICP-MS instrument at CCT mode
were as follows: RF power 1500 W, coolant gas flow 14.3 L/min,
CCT gas flow (7% H2 /He):3 ml/min; auxiliary gas flow 0.95 L/min;
nebulizer gas flow 0.87 L/min; pump rate 1.0 ml/min; peak jumping
data acquisition mode: dwell time 10 ms; uptake timings 40 s and
three replicates per sample.
For the detection of iron, the signals of 54 Fe, 58 Fe, 60 Ni, 53 Cr
and 72 Ge (IS) were monitored but only 58 Fe were used for calculations. Other elements were used to correct interference, correction
formula was as follows:
58
Fefact = Total counts at mass 58
− 0.05593 ∗ (54 Fe − 0.24921 ∗ 53 Cr) − 2.59021 ∗ 60 Ni
(1)
2.4. Sample preparation
An aliquot of 0.05 ml rat plasma was diluted to a final volume of
1.0 ml using the dilution solution containing Triton X-100 (0.01%,
v/v), nitric acid (0.05%, v/v) and N-butyl alcohol (2%, v/v). After
vortex-mixing, the aliquot was injected for analysis by ICP-MS. All
the samples were prepared using plastic pipes.
D. Zhao et al. / Journal of Pharmaceutical and Biomedical Analysis 88 (2014) 331–336
333
2.5. Method validation procedures
Full validation according to the FDA guidelines was performed
for the method.
The selectivity of the assay for 58 Fe versus endogenous substances in the matrix was assessed by comparing the scans from
six different blank rat plasma, blank rat plasma spiked with analyte
and the rat plasma sample. These samples were treated according
to the sample preparation procedure described above.
The calibration curves were prepared at different days. The
plasma samples with concentrations of 0, 0.005, 0.01, 0.02, 0.05, 0.1,
0.2, 0.8, 1.0 ␮g/ml for 58 Fe were freshly prepared with blank plasma
and working solution under the same conditions as the test samples. Modified CPS (counts per second) was used for calculations.
Quality control samples containing three different concentrations
(0.01, 0.1, 0.8 ␮g/ml) were prepared in the same way for calibration
curve samples. The low limit of quantitation (LLOQ) was calculated
as the concentration at a signal-to-noise ratio of 10:1.
For precision validation, five QC samples at three different
concentrations were evaluated for three successive days. The accuracy was determined by calculating the percentage of deviation
observed in the analysis of QC samples and expressed as the relative
error (RE%). The precision was evaluated by the relative standard
deviation (RSD%). Both RE and RSD should generally be less than
15%, and less than 20% in the vicinity of lower limit of quantification. The absolute recoveries and matrix effects for 58 Fe from
plasma were calculated at three QC levels according to the modified
equation in Erin Chambers’s research [24].
Stability experiments were performed to evaluate the stability for 58 Fe using five QC samples at three different levels in
plasma samples under different temperatures and timing conditions: short-term stability storage at room temperature for 4 h,
post-preparative stability at 4 ◦ C for 24 h, three cycles of freezethaw (room temperature) stability, and long-term stability storage
at −20 ◦ C for 15 successive days. Samples were considered to be
stable if 85–115% of the initial concentration was recovered.
2.6. Application in pharmacokinetic studies
Male and female Sprague-Dawley rats (n = 24, 200 ± 20 g) were
provided by the Shanghai Sino-British Sippr/BK LAB Animal Co.
Ltd (Shanghai, China) and certificate number was scxk (hu) 20080016. Animals were allowed to adapt to the housing environment
(20 ± 2 ◦ C, H50 ± 20%, with natural light–dark cycle) for 1 week
prior to study. The animals were fasted overnight (12 h) before the
experiment and had free access to water throughout the experimental period. All animal studies were approved by the Animal
Ethics Committee of China Pharmaceutical University.
Rats were randomly divided into two groups: i.v. and i.g., with
three sub-divisions for different dosages in i.g. group and six rats
in each group. The dosages were 2 mg/kg (equivalent to 58 Fe at
0.18 mg/kg) with the volume of 4 ml/kg for i.v. and 40, 80 and
120 mg/kg (equivalent to 58 Fe at 3.55 mg/kg) with the volume of
10 ml/kg for i.g. Blood-sampling times were arranged as 0, 0.5, 1,
1.5, 2, 4, 8, 15, 24, 36 h and 0, 0.17, 0.5, 1, 2, 4, 8, 15, 24, 36 h postdose for i.g. and i.v. groups, respectively. When each time point
reached, blood samples (about 200 ␮L) were collected from the
fossa orbitalis and centrifuged immediately at 12,000 g for 3 min.
Plasma of 50 ␮L volume was finally harvested. All samples were
stored at −20 ◦ C until analysis.
2.7. Data analysis
The pharmacokinetic parameters were calculated using
the pharmacokinetic software DAS 2.1.1 (China) by noncompartmental method.
Fig. 2. Variation of intercept, sensitivity and correlation coefficients between concentration and CPS (count for second) before and after equation editor. (a) before
equation (1). (b) after equation (1).
Absolute bioavailability (F) was determined by the equation:
F = (AUC0−∞i.g. × Dosei.v. )/(AUC0−∞i.v. × Dosei.g. )
where i.v. means intravenous administration and i.g. means intragastric administration.
All data were presented as means with their standard deviation.
Systemic exposures (AUC) were compared with a one way ANOVA.
The p value for statistical significance was set at <0.05.
3. Results and discussion
3.1. Method development
3.1.1. Compensation for interference and optimization of mass
spectrometric conditions
As one of the naturally occurring isotopes for iron, 58 Fe
(0.28%) could be used for bioavailability study and pharmacokinetic research. However, 58 Fe is not a real tracer due to the high
background in various matrixes. At the same time, 58 Ni can create
a spectral interference at the same mass as the 58 Fe which called
isobaric interferences [25].
To compensate for isobaric and background interferences, a
mathematical interference correction equation has been applied by
knowing the ratio of the intensity of the interfering species at the
analyzed mass to its intensity at the alternate mass. The correction
is made in the following manner:
Total counts at mass 58 = 58 Fefact + 58 Febackground + 58 Ni
58 Fe
Therefore
counts
at
mass
fact = total
58
58 − Febackground − 58 Ni
To find out the contribution from 58 Febackground and 58 Ni, it is
measured at the interference free isotope of 54 Fe and 60 Ni and a
correction of the ratios of 58 Fe/54 Fe and 58 Ni/60 Ni are applied as
equation:
58
Fefact = total counts at mass 58 − 0.05593
× 54 Fe − 2.59021 × 60 Ni
where 0.05593 is the ratio of the natural abundance of these two
isotopes (58 Fe/54 Fe) and 2.59021 is the ratio of (58 Ni/60 Ni). The ratio
is always constant. Actually, a correction equation for 54 Fe is also
need to compensate for isobaric interferences of 54 Cr.
So, the final mathematical interference correction equation is
like this:
58 Fe
counts
at
mass
58 − 0.05593 × (54 Fe −
fact = total
0.24921 × 53 Cr) − 2.59021 × 60 Ni
Under current conditions, as the Fig. 2 shown, optimized analytical methodology has greatly minimized the negative impact of
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D. Zhao et al. / Journal of Pharmaceutical and Biomedical Analysis 88 (2014) 331–336
Table 1
Spectral interferences in the analysis of 58 Fe by ICP-MS.
Isotope
58
Fe
Fe
53
Cr
60
Ni
54
Abundance (atom %)
Interfering species
0.28
5.80
0.095
26.23
58
Ni(67.8%),4 0 Ar + 18 O(0.2%),12 C + 46 Ti(7.9%),17 OH + 41 K(6.9%),1 H + 57 Fe(2.2%),14 N + 44 Ca(2.1%)
Cr (2.4%),40 Ar + 14 N(99.2%),14 N + 40 Ca(96.6%),17 OH + 37 Cl(24.5%),1 H + 53 Cr(9.5%)
40
Ar + 13 C(1.1%),17 OH + 36 Ar(0.3%),14 N + 39 K(92.7%),16 O + 37 Cl(24.5%),12 C + 41 K(6.8%)
1
H + 59 Co(100.0%),40 Ar + 20 Ne(90.6%),12 C + 48 Ti(72.6%),14 N + 46 Ti(7.9%),16 O + 44 Ca(2.1%)
54
Table 2
Precision and accuracy for determination of 58 Fe in rat plasma.
Intra-day (n = 5)
Compounds
58
Fe
Inter-day (n = 15)
Concentration
Concentration
Accuracy
Added (ng/mL)
10
100
800
Measured (ng/mL)
9.5 ± 0.7
102.5 ± 0.8
785.6 ± 29.8
RE (%)
5.0
2.5
1.8
interference on the determination of 58 Fe and the intercept has
dropped to 1.597 from 770.8 while the coefficient has been promoted to 0.999 from 0.990.
As shown in Table 1, there is still much spectral interference
such as ArN+ /54 Fe+ , ArC+ /53 Cr+ , ArO+ /58 Fe+ and CaO+ /58 Fe+ in the
analysis of 58 Fe by ICP-MS. Compared with standard mode, CCT
mode using 8% (v/v) H2 in He as the collision gas could greatly
improved the polyatomic ion interferences. The spectrum in the
absence or presence of 58 Fe, and the peak at 58 Fe was almost at
very low background level.
3.1.2. Selection of internal standard
In this method Ge was chosen as IS for its similar mass with iron
and the abundance is low enough to ignore in the sample solution
as one of rare element.
3.1.3. Carry-over effect
To evaluate and minimize the carry-over effect, the signals of
water following the ULOQ (upper limit of quantitation) calibration
sample were detected. A 40 s rinse time with 2% HNO3 was required
to avoid a memory effect from the preceding high concentration
sample.
3.2. Method validation
3.2.1. Selectivity and linearity
The selectivity was checked with six different blank plasma
samples. No significant interferences from the endogenous plasma
components were observed at the same mass of the analyte.
Standard curves for 58 Fe in spiked rat plasma exhibited good
linearity over the concentration ranges 0.005–1.0 ␮g/ml with the
correlation coefficients (R2 ) > 0.99.
The LLOQ for 58 Fe in spiked rat plasma was proved to be
0.005 ␮g/ml.
3.2.2. Assay precision and accuracy
Assay precision and accuracy were calculated after analysis in
three different analytical runs. The data indicated good accuracy
and precision of the method. As shown in Table 2, the accuracy
values for intra- and inter-day were all within 100 ± 10% of the
actual values at each QC level; the intra- and inter-day precisions
of the assay were below 10.86% for 58 Fe. The results indicated
that the method was reproducible. In general the accuracy (10.8%)
achieved analytically in this paper has shown to be much better
than regular researches [2] and conformed to the requirements of
FDA.
RSD (%)
Concentration
Accuracy
RSD (%)
7.7
0.8
3.8
Measured (ng/mL)
10.7 ± 1.2
107.8 ± 4.8
826.4 ± 39.0
RE (%)
6.8
7.8
3.3
10.9
4.4
4.7
3.2.3. Absolute recovery and matrix effect
The mean plasma absolute recoveries for 58 Fe at different levels
were from 87.3 to 111.3%. The overall yields were shown in Table 3
and the results showed that data were consistent and precise.
After optimization of mass spectrometric conditions and compensate for interference, no apparent ionization interference was
found in the analysis for 58 Fe. Results of matrix effect were no
less than 88.8%, showing that there was no significant difference between the CPS of samples spiked into matrix after the
pretreatment procedure and from the dilution solution. It indicated
that the interferences from matrix have been compensated well.
3.2.4. Stability
No significant reduction of 58 Fe was detected for the plasma
samples under different conditions by simulating the same conditions during sample analysis. The results were summarized in
Table 4.
3.3. Application in the pharmacokinetic study
The method was successfully applied to the pharmacokinetic
study of 58 Fe originating from hemin extrinsically labeled. Noncompartmental pharmacokinetic parameters of 58 Fe originating
from hemin extrinsically labeled in plasma after intravenous and
oral administration for rats are shown in Table 5 and concentrationtime profiles of 58 Fe in rat plasma following administrations are
presented in Fig. 3. After intragastric administration to rats, the
peak plasma concentrations were reached at about 2 h after dosing and the mean terminal half-life was 6.62 ± 1.55, 7.92 ± 2.49,
8.10 ± 2.37 h at the doses of 40, 80 and 120 mg/kg (equivalent to
58 Fe at 3.55 mg/kg), respectively. Compared with exposure of intravenous dosing group in rats, the absolute bioavailability of 58 Fe
originating from hemin extrinsically labeled was 1.07 ± 0.21% at
40 mg/kg.
Systemic exposures (AUC) for intragastric administration at different dose levels (while equivalent to 58 Fe for the same dose
at 3.55 mg/kg) were compared and as shown in Table 5, there
were significant statistically differences between the low dose
Table 3
Absolute recovery and matrix effect for determination of 58 Fe in rat plasma.
Compound
58
Fe
Concentration
(ng/mL)
Recovery (%)
Matrix effect (%)
Mean ± SD
RSD (%)
Mean ± SD
RSD (%)
10
100
800
111.3 ± 9.3
87.3 ± 3.5
96.8 ± 1.6
8.4
4.0
1.7
88.8 ± 4.8
99.0 ± 1.7
98.0 ± 3.8
5.4
1.7
3.9
D. Zhao et al. / Journal of Pharmaceutical and Biomedical Analysis 88 (2014) 331–336
335
Table 4
Stability (%) of 58 Fe in rat plasma under different conditions.
Compound
Concentration (ng/mL)
Room temperature
stability for 4 h
Post-preparative
stability at 4 ◦ C for 24 h
Freeze-thaw stability
for three cycles
Frozen stability for 15 days
58
10
100
800
102.8 ± 12.1
102.7 ± 1.6
101.8 ± 0.8
89.4 ± 11.0
100.1 ± 1.9
101.4 ± 1.1
99.9 ± 15.3
105.4 ± 1.6
101.3 ± 1.4
93.7 ± 6.1
102.1 ± 0.9
102.1 ± 0.8
Fe
Table 5
Mean pharmacokinetic parameters of
rats, respectively (mean ± S.D, n = 6).
Parameters
58
Fe in plasma after administration of hemin at different dose levels (while equivalent to
58
iron for the same dose at 3.55 mg/kg) to
i.g.
i.v.
Dose (mg/kg)
40
80
120
2
Tmax (h)
Cmax (␮g/L)
AUC0−t (␮g.h/L)
AUC0−∞ (␮g.h/L)
Vz (L/kg)
CLz (L/h/kg)
t1/2 (h)
MRT0−t (h)
Bioavalability (F, %)
2.3 ± 0.8
197.5 ± 50.0
1837.1 ± 365.4
1916.4 ± 373.2
14.7 ± 3.6
1.6 ± 0.3
6.6 ± 1.6
8.4 ± 1.7
1.1 ± 0.2
2.9 ± 0.7
141.4 ± 33.8
1032.8 ± 355.6*
1086.2 ± 365.8*
25.9 ± 14.5
2.9 ± 0.7
6.4 ± 3.3
7.9 ± 2.5
0.7 ± 0.3
1.7 ± 0.5
111.9 ± 45.1
1025.2 ± 593.5*
1122.4 ± 605.1*
37.7 ± 26.2
3.1 ± 1.3
8.1 ± 2.4
8.6 ± 1.1
0.6 ± 0.3
–
2002.1 ± 112.8
8348.9 ± 539.5
8984.7 ± 562.7
0.3 ± 0.2
0.016 ± 0.001
14.5 ± 8.0
5.9 ± 0.3
–
i.v.: intravenous administration; i.g.: intragastric administration. *p < 0.05 vs low dose group.
group and other groups. This disproportional increase at the highdose level suggested that uptake process, rather than excretion
process, are saturated. Due to the lack of observed sex-related
differences in pharmacokinetics, male and female data were combined.
Currently, there are three methods for estimation of iron absorption. One method is to study the incorporation of stable iron
isotopes into erythrocytes after isotope administration for 14 days
[26,27]. But this method can’t obtain concentration–time profiles.
At the same time due to the relatively long cycle and complex
calculation, a large individual variation was found in Fernando
Pizarro’s study, even more, the cost of experiment was relatively
high. However, this method is the most commonly used for iron
absorption study up to now. The second one is to collect and
determinate the amount of iron isotopes in excreta for healthy
subjects and iron absorption was measured by calculating difference between uptake and excretion. Huo et al. have studied the
iron absorption of NaFeEDTA in human body using this method
but similar disadvantage existed [28]. The third one is to evaluate
iron absorption by absolutely bioavailability; a common method for
pharmacokinetic study and this view was supported in Michael B’s
study [29]. However, a recent paper investigated the availability
of hemin in humans and found a bioavailability of 5% [30]. Due to
analytical problems it is very hard to find other references about
availability of hemin in human or rat, species difference should be
investigated in the future study.
In our study, isotope concentration, not ratio was treated as
information for absorption study and a relative low individual
variation can be found using a developed ICP-MS method after
extrinsically 58 Fe labeling hemin administration. Those indicated
that extrinsically label techniques in combination with ICP-MS will
become a new tool for the analysis of endogenous substances.
4. Conclusion
The ICP-MS method described above has been proved to be
sensitive, selective and rapid for determination of 58 Fe originating
from hemin in rat plasma samples. The optimized method was validated to guarantee the need of the determination. The calibration
curve was linear within the ranges from 0.005 to 1.0 ␮g/ml with
all the correlation coefficients (R2 ) > 0.99. The accuracies and precisions determined within three consecutive days were in acceptable
limits. And there was no significant matrix effect. It was successfully applied to iron absorption study of hemin extrinsically labeled
with 58 Fe after administration to rats. Those indicated that this
will be a new tool for iron absorption study and the successfully
application indicated that the information got in this paper about
absolute bioavailability and pharmacokinetic parameters would be
an acceptable reference for further study. In the future, the method
established in this paper could be used in absorption study for similar metallotherapeutic drugs including endogenous isotope such
as calcium.
Acknowledgements
Fig. 3. Concentration–time profiles of 58 Fe in rat plasma following administrations of hemin extrinsically labeled to rats. : i.g., 40 mg/kg (equivalent to 58 Fe at
3.55 mg/kg); : i.g., 80 mg/kg (equivalent to 58 Fe at 3.55 mg/kg); : i.g., 120 mg/kg
(equivalent to 58 Fe at 3.55 mg/kg); 䊉: i.v., 2 mg/kg (equivalent to 58 Fe at 0.18 mg/kg)
(mean ± S.D, n = 6).
This work was supported by Jiangsu province Nanjing City Innovative Graduate Research Program (No.CXZZ11 0828).
We appreciate the technical assistance of engineer Xin Zheng
who worked in the Thermo Fisher Scientific in the initial
experiments.
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D. Zhao et al. / Journal of Pharmaceutical and Biomedical Analysis 88 (2014) 331–336
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