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Simultaneous Determination of Essential Basic Amino Acids in Pharmaceuticals by Capillary Isotachophoresis.

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Arch. Pharm. Chem. Life Sci. 2006, 339, 96 – 99
Short Communication
Simultaneous Determination of Essential Basic Amino Acids
in Pharmaceuticals by Capillary Isotachophoresis
Peter Kubačk, Peter MikuÐ, Iva ValÐkov, Emil Havrnek
Department of Pharmaceutical Analysis and Nuclear Pharmacy, Faculty of Pharmacy, Comenius University,
Bratislava, Slovak Republic
Capillary isotachophoresis (ITP) in cationic regime of the separation with conductometric detection has been used for the separation and determination of basic amino acids (arginine, histidine, and lysine) in pharmaceutical preparations. Several electrolyte systems of different compositions and pH were examined. The optimized ITP electrolyte system consisted of 10 mmol/L
potassium acetate adjusted to pH 4.0 with acetic acid as the leading electrolyte with the electroosmotic-flow (EOF) suppressing additive, 0.2% (w/v) methylhydroxyethylcellulose (m-HEC),
and 10 mmol/L b-alanine as the terminating electrolyte. The proposed electrophoretic method
was successfully validated. It was convenient for the sensitive, simple, rapid and highly reproducible assay of these amino acids. Good quantitation was obtained in short analysis times (a single
analysis took about 10 min). The minimal sample pretreatment and low running costs make the
proposed ITP method a good alternative to commonly used analytical methods. The obtained
results suggest that the proposed method is suitable for routine assay of basic amino acids in
Keywords: Basic amino acids / Drug / Pharmaceutical analysis / Capillary isotachophoresis / Capillary electrophoresis /
Received: May 16, 2005; Accepted: September 19, 2005
DOI 10.1002/ardp.200500131
Basic amino acids (arginine, histidine, lysine) (Figure 1)
fall into the class of twenty proteinogenic amino acids
and for mammals they are essential. L-arginine (ARG) [2amino-5-guanidinopentanoic acid] is an amino acid that
plays a role in cell division, healing of wounds, removing
ammonia from the body, improving immunity to illness,
and hormone secretion. L-histidine (HIS) [2-amino-3-(4imidazolyl)propionic acid] is metabolized into the neurotransmitter histamine and, in the stomach, it is also helpful in producing gastric juices. L-lysine (LYS) [2,6-diaminohexanoic acid] assists in calcium absorption and maintaining the correct nitrogen balance in the body. Furthermore, it is needed to produce antibodies, hormones,
Correspondence: Dr. Peter Kubačk, Department of Pharmaceutical
Analysis and Nuclear Pharmacy, Faculty of Pharmacy, Comenius University, Odbojrov 10, SK-832 32 Bratislava, Slovak Republic.
Fax: +42 1250117-100
2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Chemical structures of Arginine, Histidine, and Lysine.
enzymes, collagen formation as well as for the repair of
tissue [1].
A variety of methods have been reported for the simultaneous determination of ARG, HIS, LYS presented in substances, pharmaceutical formulations, or biological
fluids. These methods include capillary electrophoresis
(CE) [2–9], capillary electrophoresis coupled to electrospray ionization mass spectrometry [10], micellar electrokinetic capillary chromatography [11], high performance
liquid chromatography [12–16], ion exchange chromatography [17, 18], gas chromatography [19], gas chromatography-mass spectrometry [20], and stripping square
wave voltammetry [21]. The official method presented in
Arch. Pharm. Chem. Life Sci. 2006, 339, 96 – 99
the European Pharmacopoeia for their pure form assay
involves titrimetry with hydrochloric acid using methyl
red mixed solution as indicator (ARG) and potentiometric titrimetry with hydrochloric acid (HIS) or perchloric acid (LYS) [22].
Capillary isotachophoresis (ITP) is an electrophoretic
separation technique in a discontinuous buffer system in
which the analytes migrate according to their electrophoretic mobilities, forming a chain of adjacent zones
moving with equal velocity between two solutions, leading (LE) and terminating (TE) electrolyt, bracketing the
mobility range of the analytes. The major advantage of
this method is that analytes cannot break out of their
zone with the net effect that the zone compresses and
the concentration of the analyte is increased [23]. ITP is a
simple, quick and low-cost method and therefore well
suited for main drug determination. ITP was applied for
the separation of mixtures of amino acids after their derivatization with citraconic anhydride [24]. ITP was used
also for the determination of neurotoxic pyroglutamic
acid in parenteral amino acid solutions [25]. The aim of
this work was to optimize conditions for ITP method
with conductometric detection for the assay of essential
basic amino acids (ARG, HIS and LYS) in their native
forms present in pharmaceutical preparations (infusions).
Results and discussion
Method optimization
ARG, HIS and LYS have basic side chains and are well soluble in water. Therefore, they are suitable for direct cationic ITP analysis with a minimal sample pretreatment.
The principal operating parameters optimized in the present ITP separation were electrolyte systems composition, including type and concentration of leading cation,
counterion and terminating cation and pH of the leading
electrolyte. The experiments revealed the strong influence of migration parameters (migration velocity, separation efficiency) of ARG, HIS, and LYS by pH of the leading electrolyte.
Electrolyte systems with K+ as the leading ion and acetate as the counter ion were tested in the pH range from
3.5 to 5.5. b-alanine was used as the terminating ion. It
was found that at lower pH (pH a 3.5) ARG, HIS, and LYS
migrated in a mixed zone with the terminator. At higher
pH (pH >4.5) all analytes migrated separately from the
terminator but together in a mixed zone (Figure 2). The
optimized ITP separation conditions consisted of
10 mmol/L potassium acetate adjusted to pH 4.0 with
acetic acid (leading electrolyte) and 10 mmol/L b-alanine
2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Capillary isotachophoresis of essential basic amino acids
Figure 2. pH influence of ITP seperation of ARG, HIS, and LYS
in model mixture.
(terminating electrolyte). An electroosmotic flow (EOF)
was eliminated by a dynamic coating of the inner surface
of the capillary. A polymeric additive served for this purpose, methylhydroxyethylcellulose (m-HEC), present in
the leading electrolyte at a concentration of 0.2% (w/v).
The driving current applied to the capillary was 250 lA
and a single analysis took about 10 min.
After optimization of the separation conditions, some
analytical characteristics of the developed ITP method
were investigated using standard solutions, as given in
the Experimental section. The validation parameters
included sensitivity, linearity, precision (run-to-run, dayto-day), and accuracy.
The linearity was tested from 10.0 to 100.0 mg/L of
each compound, with a determination coefficient (r2) of
0.999. The equation for calibration curve is: y = a + bx,
where y is the zone length in seconds and x is the concentration (mg/L). Calibration data are given in Table 1. Relatively low values of the intercept a are a positive sign of
correctly passed migration and analytical stability of the
ITP zone, and the high values of the correlation coefficient confirms good rectilinearity of the calibration
The method was validated by evaluation of run-to-run
and day-to-day precision. The relative standard deviations
(RSD) of six replicate analyses of standard solutions (mixture of ARG, HIS, and LYS, each at a concentration of
50 mg/L) were 1.18% for ARG, 0.82% for HIS, and 1.24%
for LYS, indicating good run-to-run precision of the ITP
method. Day-to-day reproducibility of the calibration
curves was characterized by the RSD of the slopes and
P. Kubačk et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 96 – 99
Table 1. ITP results for RSHa) reproducibility and calibration.
R.S.D. [%]; (n = 5)
Range [mg/L]
Intercept (a)
Slope (b)
r2 c)
Relative step heights, relative position of the analyte zone
towards zones of leading and terminating ions measured via
changes of conductivity.
Standard deviation values of intercept (Sa) and slope (Sb).
Coefficient of determination.
intercept data not exceeding 1.86%. The limit of detection (estimated as 3r) and limit of quantitation (estimated as 10r) values for each compound were 4 mg/L
and 10 mg/L, respectively. The sensitivity of this proposed
method was sufficient for the assay of ARG, HIS, and LYS
in pharmaceuticals.
The validated method was successfully applied to the
determination of ARG, HIS, and LYS in commercial pharmaceutical formulations (infusions), as shown in Table 2.
The contents of these amino acids obtained by the proposed method were in good agreements with those
declared by the manufacturers. Quantitative estimations
of the drug brought consistent results and the absolute
differences between determined and declared values ranged from 0.1 to 2.0%.
The results from the recovery test indicated a practical
use of the proposed method. The method accuracy was
examined by adding a known amount of standard of
ARG, HIS, and LYS to the real samples (infusions). Concentration of added standards was 50 mg/L and the recoveries were found to be 98.51–99.28%. Hence, it can be concluded that the accuracy of the proposed ITP assay of
basic amino acids in infusions is acceptable.
Isotachopherogram from the ITP separation of ARG,
HIS, and LYS, present in the real sample (Aminoplasmal
5% E), is presented in Figure 3. No interference from the
Figure 3. Isotachopherogram from the ITP separation of ARG,
HIS, and LYS in infusion Aminoplasmal 5% E.
sample solvent and dosage form excipients could be
observed by analyzing the samples.
The advantages of the proposed method are short analysis time, low consumption of electrolytes and samples,
and direct injection of samples without their pretreatment. Performance parameters including precision
(repeatability, intermediate precision, reproducibility)
and accuracy are comparable or better than those
obtained by alternative methods (Capillary zone electrophoresis (CZE), HPLC) [4, 8]. We conclude that the
reported operating conditions are suitable for the routine assay of basic amino acids present in pharmaceuticals. The proposed ITP method should be convenient also
for complex biological sample applications if employed
as purification and pre-concentration CE stage in an online coupled ITP-CZE method.
Table 2. Quantification of basic amino acids in infusions.
Aminoplasmalm 5% Ea)
Neonutrinm 5%a)
Nutramin Nm 8%a)
Mean [%]
RSD [%]; (n = 6)
ARG, HIS, and LYS were determined in three different batches of the preparation.
2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Arch. Pharm. Chem. Life Sci. 2006, 339, 96 – 99
This work was supported by grants from the Slovak Grant Agency for Science under the projects No. 1/1196/04 and 1/2310/05.
Capillary isotachophoresis of essential basic amino acids
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Slovak Republic) was used in a single-column configuration of
the separation unit. The analyzer was equipped with a 30 lL
sampling valve, a 16060.8 mm (i. d.) separation capillary made
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Chemicals and samples
Chemicals used for the preparations of the electrolyte solutions
were obtained from Merck (Darmstadt, Germany) and Aldrich
(Steinheim, Germany). The water was dematerialized by a Rowapure-Ultrapure water purification system (Premier, Phoenix, AZ,
USA.). All chemicals used were of analytical grade or were additionally purified by the usual methods. Acetic acid was used for
the preparation of the electrolyte solutions after isothermal distillation. Methylhydroxyethylcellulose 30 000 (m-HEC) was
obtained from Serva (Heidelberg, Germany). The electrolyte solutions were filtered before use through disposable membrane filters (1.2 lm pore size) purchased from Sigma (St. Louis, MO,
USA). The standards of ARG, HIS, and LYS were obtained from MP
Biomedicals (Aurora, OH, USA). The analyzed infusions Aminoplasmal 5% E (B. Braun AG, Melsungen, Germany), Nutramin N
8%, and Neonutrin 5% (both Infusia a.s., Horatev, Czech Republic) were obtained commercially. These infusions are mixtures of
about 20 amino acids used for supplementary or complete parenteral nutrition.
Sample and standard solution preparations
The infusions Aminoplasmal 5% E (containing 4.6 g/L of ARG, 2.6
g/L of HIS, and 3.5 g/L of LYS), Nutramin N 8% (9.1 g/L of ARG, 2.8
g/L of HIS, and 5.4 g/L of LYS) and Neonutrin 5% (4.5 g/L of ARG,
1.75 g/L of HIS, and 4.05 g/L of LYS) were diluted with dematerialized water in ratio 1:100 prior to the analyses and directly
injected into the ITP equipment. The pure standard stock solution of a mixture of ARG, HIS, and LYS (conc. 400 mg/L each) was
prepared by dissolving these amino acids in dematerialized
water. Prior to the analysis, it was appropriately diluted with the
water to the final concentration of 50 mg/L for all analytes and
directly injected.
2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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