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Validation of HPLC method for analysis of adenine in plasma

Conference Paper · July 2012

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Analytical
Methods
PAPER

Fast HPLC analysis of adenine in human plasma using a

new generation C28 column and different extraction
Cite this: Anal. Methods, 2013, 5, 1487
methods
Mohammed Al Za'abi,a Badreldin H. Ali,a Afzal Hussaina and Imran Ali*b

A fast, simple, inexpensive, selective, rugged and reproducible HPLC method based on the use of a new
generation Sunniest RP-Aqua C28 column is described for the analysis of adenine in plasma. Three
methods for the extraction of adenine from human plasma i.e. by acetone, acetonitrile and solid phase
cartridges are reported. The separation was performed via isocratic elution of adenine using water–
acetonitrile (90 : 10, v/v) as mobile phase at a flow rate of 1.0 mL min
1 and with detection at 260 nm.
The values of retention factor in standard and plasma samples were 2.53 and 2.60, respectively. The
Received 9th November 2012
Accepted 26th December 2012
method was validated and found to be linear at a concentration range of 5–1000 mg mL
1. The limits of
detection and quantification were lower than 0.3 and 0.91 mg mL
1, respectively. The intra- and inter-
DOI: 10.1039/c2ay26363g
day precisions (% RSD) were less than 9%. The accuracy values were between 99.8 and 107.4%. The
www.rsc.org/methods extraction recoveries ranged from 95–80%.

Introduction weaknesses of sample preparation methods restrict their use in

routine clinical analyses of adenine. Moreover, the use of a C18
Adenine, a purine nucleobase (C5H5N5, Fig. 1) is an integral column could not provide sharp and fast separation. But
component of nucleic acids and adenosine triphosphate (ATP).1 recently, a new generation column namely the C28 column has
It is metabolized by an enzyme xanthine dehydrogenase to 2,8- been marketed, which seems to be better than the C18 column.
dihydroxyadenine (DHA) in animals including human beings.2 The analysis of adenine in plasma is a tedious job involving
Both adenine and DHA precipitate and form crystals in the rigorous sample preparation due to the presence of thousands
renal tubules because of their low solubility.3 Thus, long oral of other entities.26 The sample preparation is one of the most
consumption of adenine may cause the occlusion of renal important parts in analytical science for monitoring drugs in
tubules. Consequently, it retards the excretion of nitrogenous different biological samples. About 80 percent of analytical
substances; leading to a status resembling chronic renal failure chemists use solid phase extraction (SPE) as the method of
in human beings.4 Therefore, adenine is becoming a focus of choice for sample preparation in human plasma.27 But, some-
research in acute and chronic kidney failure and other clinical times, SPE is also not capable of removing all of the impurities
conditions.4,5 Thus, adenine has been mixed with feed and from plasma. A method for the measurement of adenine in
given to animal models to induce acute and chronic renal plasma for the concentration–effect relationship and to estab-
failure.6,7 These experiments need an accurate method for the lish pharmacokinetic proles is warranted. Therefore, different
monitoring of adenine in animal model plasma. Among various sample preparation methods were explored for the extraction of
methods, HPLC is supposed to be the best technique due to its adenine from human plasma. Attempts have also been made to
efficiency, selectivity, rapidness, reproducibility, and to the low develop and validate a fast, selective, effective and reproducible
detection limits obtained. A literature survey indicates that few HPLC method for the analysis of adenine in human plasma.
papers have been published on HPLC analyses of adenine in The results of these ndings are given herein.
plasma.8–11 Besides, many research papers are available for the
determination of adenine derivatives such as adenine nucleo- Experimental
tides.12–25 Furthermore, it was observed that all the methods
published have some drawbacks such as high limit of detection, Materials and reagents
high run time and use of costly solvents. Besides, certain Adenine was obtained from Sigma (St Louis, USA). Acetonitrile
and methanol (HPLC grade) were purchased from J.T. Baker
a
(Deventer, Holland). C18 solid phase cartridges were purchased
Department of Pharmacology and Clinical Pharmacy, College of Medicine and Health
Sciences, Sultan Qaboos University, Muscat, Sultanate of Oman from Waters, USA. The centrifuge machine used was from
b
Department of Chemistry, Jamia Millia Islamia (Central University), New Delhi, India. Eppendorf (model 5415 R) Germany. Pure water was obtained
E-mail: drimran_ali@yahoo.com; drimran.chiral@gmail.com using a Milli-Q system (Millipore, Milford, MA).

This journal is ª The Royal Society of Chemistry 2013 Anal. Methods, 2013, 5, 1487–1493 | 1487
Analytical Methods Paper

the standard method.28,29 The plasma samples were extracted by

three methods as described below:
Extraction with acetonitrile. Plasma sample (250 mL) was
pipetted into a polypropylene tube and 1.0 mL acetonitrile was
added followed by vortex mixing for 30 s. The tube was centri-
fuged for 5.0 min at 5000g and the resultant supernatant was
evaporated under gentle nitrogen pressure. HPLC mobile phase
[water–acetonitrile (90 : 10, v/v); 0.5 mL] was added and an
aliquot (5.0 mL) was injected into the chromatographic system.
Extraction with acetone. Plasma sample (250 mL) was pipet-
ted into a polypropylene tube and 1.0 mL acetone was added
followed by vortex mixing for 30 s. The tube was then centri-
fuged for 5.0 min at 10 000 rpm and the resultant supernatant
was evaporated under gentle nitrogen pressure. The residue was
re-dissolved in 0.5 mL of mobile phase. An aliquot of 5.0 mL was
injected into the chromatographic system as were standard
Fig. 1 Chemical structure of adenine. solutions.
Extraction with solid phase cartridge. To study the interac-
Stock solutions and standards tions of adenine with plasma, 1.0 mL adenine solution (0.1 mg
mL
1) was mixed with 1.0 mL plasma and vortexed for 5.0 min
Stock solution was prepared by dissolving adenine (1.0 mg
followed by 10 min shaking. Aer settlement for 1.0 h, 15.0 mL
mL
1) in Millipore water. Further standard solutions were
acetone was added. The mixture was then centrifuged at 3000
obtained by serial dilutions of the stock solution with Millipore
rpm for 10 min and the supernatant was collected and evapo-
water and were stored at
20  C. These samples were stable for
rated to dryness. The residue was reconstituted with 5.0 mL
5 weeks. The calibration and quality control plasma samples
phosphate buffer (100 nM, pH 8.0). A C18 solid phase cartridge
were prepared by addition of standard solutions to adenine free
(Waters, USA) was used for extraction. The cartridge was pre-
plasma.
conditioned with 2.0 mL MeOH followed by 5.0 mL Millipore
water. Spiked sample was passed through the cartridge at a ow
Sample preparation and extraction rate of 0.5 mL min
1 followed by cartridge washing with 2.0 mL
The plasma samples were stored at
20  C freezer and thawed water at the same ow rate. The cartridge was then dried by
at room temperature before processing. First, to exploit the passing hot air and adenine was eluted with 5.0 mL methanol
experimental errors, blank experiments were carried out as per containing 0.1% acetic acid at a ow rate of 0.5 mL min
1. The

Fig. 2 Percentage recoveries of adenine from human plasma and the chromatograms showing impurities and peaks, (A): extraction with acetone, (B): extraction with
acetonitrile and (C): extraction with solid phase cartridge.

1488 | Anal. Methods, 2013, 5, 1487–1493 This journal is ª The Royal Society of Chemistry 2013
Paper Analytical Methods

Fig. 3 HPLC chromatogram of adenine (500 mg mL
1) in water after extraction with acetonitrile (standard).

Fig. 4 HPLC chromatogram of plasma spiked adenine (500 mg mL
1) after extraction with acetonitrile.

This journal is ª The Royal Society of Chemistry 2013 Anal. Methods, 2013, 5, 1487–1493 | 1489
Analytical Methods Paper

used was water–acetonitrile (90 : 10, v/v) at a ow rate of 1.0 mL

min
1. The detection wavelength was set at 260 nm.

Validation procedures
Linearity. The selectivity of the method was assessed by
comparing chromatograms of standard solutions. The linearity
of the method was evaluated by constructing calibration curves
in the range of 5–1000 mg mL
1, (5, 10, 25, 50, 100, 500 and
1000 mg mL
1 of adenine). The calibration curves were gener-
ated by linear regression analysis of the peak areas against the
nominal concentration. The calibration curve was used to
calculate the concentrations of adenine in plasma.
Accuracy and precision. The intra- and inter-day precision
and accuracy were investigated by HPLC analysis of three
Fig. 5 Effect of acetonitrile on separation factor of adenine. different sample concentrations (7, 35 and 850 mg mL
1) over
four consecutive days. The samples of three different concen-
trations were tested in six replicates and calculated with cali-
Table 1 Accuracy, intra- and inter-day precisions of adenine in plasmaa bration curves obtained daily. Accuracy was expressed as
percentage value [% accuracy ¼ (detected concentration/
Intra-day (n ¼ 6) Inter-day (n ¼ 12)
nominal concentration)  100%]. The precision was estimated
Concentrations/ Accuracy Accuracy as percentage relative standard deviation (RSD). For acceptable
mg mL
1 (%) RSD (%) (%) RSD (%) intra- and inter-day values, accuracy and precision should be
85–115% and be #15%, respectively; except for limit of quan-
7.0 104.2 5.5 102.6 8.6
35.0 107.4 3.7 102.9 3.0 tication (LOQ), where accuracy should be between 80–120%
850.0 101.3 2.4 99.8 2.1 with precision not exceeding 20%.30
a LOD and LOQ. The combined standard solutions were
RSD: relative standard deviation expressed as CV (%).
prepared by sequential dilutions of adenine and injected at
lower concentrations. The limit of detection (LOD) and LOQ for
the assay were calculated by using the signal-to-noise ratio.30,31
eluted methanol was concentrated under vacuum to 0.5 mL.
LOD and LOQ were determined when the signal-to-noise ratio
Further, it was ltered through a 0.22 mm membrane and used
were at least three and ve times, respectively.
for HPLC analysis.
Robustness. For the determination of method robustness, a
slight variation in the chromatographic parameters such as ow
Liquid chromatographic conditions rate, temperature, mobile phase composition and wavelength
HPLC was performed with a Waters HPLC system series was carried out. The sample solutions were prepared and the
2695 equipped with an auto-sampler, empower soware data retention time, peak area and shape were analyzed under the
station, version 2.0.7 and a photodiode array detector (2996 established and slightly varied experimental conditions.
module). The chromatographic separation was performed on a Stability. The stability of the samples at three concentrations
Sunniest RP-Aqua C28 column (250 mm  4.6 mm, 5 mm, was also investigated. It included (i) stability of the extracted
Chromanik, Japan) at room temperature. The mobile phase samples at room temperature for 24 h; (ii) stability aer three

Table 2 Stability results, expressed as percentages of freshly prepared quality control samples of adenine (n ¼ 4)

Short term study Long term study Freeze-thawed study

Freshly
Theoretical prepared
concentration/mg (mean  Aer 24 h storage Freshly prepared Aer 3 weeks storage at Freshly prepared Aer 3 freeze thawed
mL
1 SD) (mean  SD)a (%) (mean  SD)
20  C (mean  SD)a (%) (mean  SD) cycles (mean  SD)a (%)

7.0 7.23  6.32  0.86 (87.5) 7.15  0.82 6.43  0.83 (91.5) 7.60  0.21 7.18  0.46 (94.4)
0.43
35.0 36.0  34.4  0.60 (95.5) 36.3  0.28 35.7  1.66 (98.4) 35.6  1.22 34.6  2.04 (97.2)
0.84
850.0 859.5  837.8  29.3 (97.5) 860.0  3.62 839.8  2.5 (97.7) 850.6  9.33 829.9  26.7 (97.6)
1.43
a
Percentage calculated from freshly prepared samples.

1490 | Anal. Methods, 2013, 5, 1487–1493 This journal is ª The Royal Society of Chemistry 2013
Paper Analytical Methods

Table 3 A comparison of analyses of adenine by different HPLC/LC–MS methodsa

HPLC separation
time/min Columns used Extraction procedure Detection limit Economy Ref.

6.5 C18 SPE NG Highly expensive due to high 8

consumption of time, chemical,
electricity and man power
15.0 C18 SPE NG Highly expensive due to high 9
consumption of time, chemical,
electricity and man power
25.0 C18 SPE 1.75 mg L
1 Fairly expensive due to moderate 10
consumption of time, chemical,
electricity and man power
8.0 C18 SPE 0.50 to 1.5 ng mL
1 Very expensive due to costly LC–MS 11
instrument, moderate consumption of
time, chemical, electricity and man
power
4.0 C28 Liquid liquid extraction; 0.3 mg mL
1 Inexpensive due to low consumption of Present work
10 min time, chemical and man power
a
Briey, the present work is better than previously reported ones in terms of extraction (three types of methods), economy and limits of detection.
NG: Not given.

freeze thawed cycles with a frozen temperature of
20  C and HPLC analysis
thawing temperature of 25  C; and (iii) stability of plasma
Details of the column and mobile phase used for the HPLC
samples at
20  C for three weeks. All the samples were
analysis of adenine are given above. The chromatogram of
analyzed together with freshly processed samples.
adenine in plasma sample was identied by comparing with the
chromatogram of a standard (water) obtained under identical
chromatographic conditions. The capacity factors (k) for
Results and discussion adenine in standard sample and plasma were calculated. The
values for k were 2.53 and 2.60 for blank and plasma samples
The results and discussion of this work is divided into two parts respectively. However, the peak was slightly broad in plasma
i.e. sample preparation and HPLC analysis. These are discussed sample, which may be due to interference from plasma impu-
in the following sub-sections: rities. The chromatograms of adenine in standard solution
(water) and plasma are given in Fig. 3 and 4, respectively, and
indicate fast and baseline separation. The retention times of
Sample preparations adenine in standard and plasma samples were 3.904 and
3.971 min, respectively. A slight late elution in plasma may be
As discussed above the sample preparation was optimized by
again due to interference from some impurities in the extracted
using three methods viz. extraction with acetone, acetonitrile
plasma samples.
and solid phase cartridge. The percentage recoveries of adenine
for the different methods were calculated by considering the
experimental error. The experimental error was determined by Optimization of HPLC method
running blank experiments. The calculated percentage recov- To optimize the chromatographic conditions, various combi-
eries were 95, 90 and 80 for acetone, acetonitrile and solid phase nations of acetonitrile and water were tried. Besides, water and
cartridge, respectively. It was observed that the acetone extracts methanol mixtures, various buffers were also used to achieve
contained more impurities than the acetonitrile extracts (Fig. 2). the best separation. As a result of exhaustive experimentation
Therefore, acetonitrile was considered to be a better extraction the best solvent system was optimized and reported. The effect
solvent than acetone. On the other hand, a higher percentage of different concentrations of acetonitrile was also studied and
recovery (90%) was obtained with acetonitrile than with the SPE shown in Fig. 5. A perusal of this gure indicates that a high
method (80%) (Fig. 2). It is also clear from Fig. 2 that acetone concentration of acetonitrile resulted in poor separation and
was used for elution from the solid phase cartridge. Aer retention factor. The values of the retention factor were 3.0 and
passing through the cartridge, the impurities present in the 2.56 for 5.0 and 10.0 mL acetonitrile, respectively, indicating
extract of acetone could not be removed; indicating the inca- good resolution but the peak was broad with 5.0 mL acetoni-
pability of the solid phase cartridge. Moreover, some adenine trile. In contrast, the values of the retention factors were 0.9, 0.4
was adsorbed on the C18 material of the solid phase cartridge; and 0.05 for 15, 20 and 25 mL acetonitrile, respectively, indi-
resulting in poor percentage recovery (80%). Finally, the aceto- cating incomplete separation. Therefore, 10.0 mL acetonitrile
nitrile method was selected as the best one and was used was found to be the best concentration in the reported mobile
throughout this study. phase.

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Analytical Methods Paper

Validation of HPLC method developed countries. Therefore, the reported method is

economical in terms of time, consumption of costly solvents
Linearity. The extraction procedure and HPLC conditions
and the man power required. Additionally, in the reported
resulted in clear and good separation between adenine and
paper the release of hazardous chemicals into the environment
endogenous interferences from plasma. The generated cali-
is low making the HPLC method eco-friendly in nature. The
bration curves, calculated using peak areas at six different
detection limit is 0.3 mg mL
1, which is better than the reported
concentrations in the range of 5–1000 mg mL
1 (5, 10, 25, 50,
methods, with the exception of the LC–MS method. Briey, the
100, 500 and 1000 mg mL
1 of adenine), were linear with an r2 >
0.999. The deviations of standards from nominal concentra- present HPLC method is better than the earlier reported ones.
tions were all within 7%. The representative chromatograms
obtained from standard and plasma spiked samples are shown Application of the developed methodology
in Fig. 3 and 4. The developed extraction and HPLC procedures were applied to
Accuracy and precision. The intra- and inter-day accuracy determine adenine in real samples. The plasma of rat, aer
and precision data were calculated for three different adenine administration of adenine, was processed as mentioned earlier.
concentrations (7.0, 35.0 and 850.0 mg mL
1). Overall values of The extracted plasma sample was analysed using the developed
RSD and accuracy were 2.4–5.5 and 101.3–107.4%, and are given HPLC method. The retention time for the adenine peak was
in Table 1. These values suggest that the reported method is 3.970 min, which was close to the standard HPLC run. There
accurate and precise. were no other peaks present in the chromatogram; indicating
LOD and LOQ. The LOD and LOQ were calculated from the the selective and efficient nature of the developed sample
calibration graph of adenine as three and ve times the signal- extraction and HPLC methods.
to-noise level for LOD and LOQ, respectively. The LOD and LOQ
values of adenine were 0.3 and 0.91 mg mL
1, respectively.
Robustness. The method robustness was evaluated by a Conclusion
slight variation in the chromatographic conditions. Under the
A fast, simple, inexpensive, selective, rugged and reproducible
slightly varied conditions there was no change in the retention
HPLC method was developed based on the use of a new
times, peak areas and symmetry. The optimum wavelength for
generation C28 column. Three extraction methods for adenine
detection and quantication was 260 nm, at which good
from human plasma were also reported; with acetonitrile being
detector response for adenine was obtained.
the best one. The percentage recoveries of adenine from plasma
Specicity. The specicity of the method was determined by
were 80 to 95%, which are quite satisfactory. The accuracy and
observing any interference from plasma. There was no inter-
precisions of the method were within the laboratory limits
ference from the plasma using the HPLC method. These results
required for biological analytical assays. The method is sensi-
indicated that the HPLC method was specic.
tive with simple and short laboratory work up time, allowing the
Stability. The stability results at short term, long term and
analysis of many samples per day. Therefore, the developed and
freeze thawed studies are given in Table 2. These results indi-
validated sample preparation and HPLC methods can be used
cated that adenine was stable aer extraction from the plasma
for monitoring adenine in plasma from any species.
stored at room temperature for 24 h and from the plasma stored
at
20  C for three weeks. Furthermore, it was also found to be
stable aer three freeze–thaw cycles. There were no signicant Acknowledgements
deviation between samples stored under the tested conditions
The authors are thankful to Sultan Qaboos University, Muscat,
and the freshly prepared ones. Overall, the values given in Table
Oman for nancial assistance.
2 indicate that adenine was quite stable under the experimental
conditions.
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