Journal of Chromatography A, 890 (2000) 273–280

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Validation of an analysis method for 4-amino-3-hydroxybutyric acid

by reversed-phase liquid chromatography
M. Candela, A. Ruiz*, F.J. Feo
´
FAES, S. A. Department of Quality Control, Maximo Aguirre 14, E-48940 Lamiaco-Lejona, Spain

Received 5 November 1999; received in revised form 17 May 2000; accepted 22 May 2000

Abstract

A rapid and simple reversed-phase liquid chromatographic method that did not require the derivatization of 4-amino-3-
hydroxybutyric acid (GABOB) was developed and validated. The method proved to be suitable for the determination of
GABOB concentrations in finished pharmaceutical product (tablets). The method was developed using a RP-18 column, UV
detection at 210 nm and 0.01 M sodium heptasulphonate solution, at pH 2.4, as the mobile phase. Different validation
parameters were included and satisfactorily evaluated. The specificity of the method was demonstrated. Linearity was
established in the range 0.40–0.60 mg / ml (r50.997). The method showed excellent accuracy (100.1%). Precision
(repeatability) gave a relative standard deviation value of 0.68%, while the intermediate precision was 1.70%. A robustness
test showing the influence of different pH values and counter-ion concentrations was also performed.  2000 Elsevier
Science B.V. All rights reserved.

Keywords: 4-Amino-3-hydroxybutyric acid

1. Introduction when they are developed and intended to be for

routine use.
One essential part of Good Manufacturing Prac- 4-Amino-3-hydroxybutyric, or g-amino-b-hydro-
tices in the pharmaceutical industry is analytical xybutyric acid (GABOB), is an amino acid used
method validation. It is the process for establishing for treating hemiplegia, memory and speech distur-
that performance characteristics of the method, ex- bances, essential neurogenous hypertension, epi-
pressed in terms of analytical parameters, are suit- lepsy and arrested mental development. Reversed-
able for the intended application. Chromatographic phase chromatographic analysis of amino acids
methods play an important role in the pharmaceu- usually includes a prior derivatization process with
tical field and, hence, they need to be validated different derivatization agents (e.g., phenylisothio-
cyanate) [1–5]. These determinations are tedious
and time-consuming. No high-performance liquid
*Corresponding author. Tel.:134-944-639-000; fax: 134-944- chromatographic (HPLC) method without derivati-
800-547. zation for GABOB determination has been re-
E-mail address: aruiz@faes.es (A. Ruiz). ported.

0021-9673 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved.
PII: S0021-9673( 00 )00605-1
274 M. Candela et al. / J. Chromatogr. A 890 (2000) 273 – 280

The aim of this work was to develop and validate hydantoin) was from Recordati (Milan, Italy), mi-
a new, simple and rapid method for the quantitative crocrystalline cellulose M-101, was from Mingtai
analysis of GABOB in a finished pharmaceutical Chemicals (Taipei, Taiwan) and magnesium stearate
product by reversed-phase chromatography. was from Mallinckrodt (St. Louis, MO, USA). HPLC
ultrapure water was generated by a Sation 9000
System Aqualab (Barcelona, Spain). The mobile
2. Experimental phase was filtered through a 0.45-mm membrane
(Millipore, Bedford, MA, USA). All samples were
2.1. Instrumental and operating conditions filtered through a hydrophilic Durapore-PVDF mem-
brane of 0.45 mm pore size (4 mm Millex-HV,
The HPLC analysis was carried out on a Merck– Millipore, Yonezawa, Japan) before injection.
Hitachi La Chrom  liquid chromatograph with an
autosampler Model L-7200 and an L-7100 pump
connected to a photodiode-array detector, L-7450A 2.3. Sample preparation
(Merck–Hitachi, Darmstadt, Germany). The column
used was a reversed-phase C 18 Purospher (2534 mm
2.3.1. Preparation of stock standard solution
I.D.; 5 mm; Merck, Darmstadt, Germany), thermo-
A 250-mg quantity of DL-g-amino-b-hydroxy-
stated at 408C. The mobile-phase composition was
butyric acid was accurately weighed and dissolved in
0.01 M sodium heptanesulphonate, at pH 2.4, with
25 ml of water, followed by the addition of mobile
H 3 PO 4 and the flow-rate was set at 1.0 ml / min. The
phase to 50 ml.
injection volume was 50 ml and the detection
wavelength was set at 210 nm. The instrument and
chromatographic data were fully managed by the 2.3.2. Preparation of standard solutions
D-7000 HPLC System Manager Software (Merck– Standard solutions were prepared by the dilution
Hitachi). of the stock standard solution with mobile phase to
A Perkin-Elmer high-performance liquid five different concentrations over the range of inter-
chromatograph equipped with an autosampler (model est, in this case, 0.40, 0.45, 0.50, 0.55 and 0.60
ISS 200), a Series 200 LC quaternary pump and an mg / ml, corresponding to 80, 90, 100, 110 and 120%
LC-235 diode array detectorn operating at 210 nmn of labelled concentration, respectively. A concen-
was used for the study of the intermediate precision. tration of 0.50 mg / ml is considered the labelled
All of the equipment’s functions and data obtained concentration, being the GABOB concentration ob-
were managed by the Turbochrom Workstation pro- tained after the extraction procedure from the tablets
gram (PE Nelson). Column, mobile phase, flow-rate, (one tablet in 200 ml of water was mechanically
injection volume and detection wavelength were the shaken for 30 min and sonicated in a water bath for 5
same as the chromatographic conditions used in the min, to make sure of its total dissolution).
Merck–Hitachi HPLC system.

2.2. Reagents and materials 2.3.3. Preparation of standard solution with

placebo
DL-g-Amino-b-hydroxybutyric acid (Sigma, St. Placebo formulation was added to the different
Louis, MO, USA) was used to prepare the standard standard solutions in an equivalent amount to the one
solutions. Phosphoric acid (85%) was purchased obtained by dissolving one tablet in 200 ml of water.
from Merck and sodium heptanesulphonate (HPLC Standard solutions with placebo samples must be
grade) was from Scharlau (Barcelona, Spain). 5- filtered through 0.45 mm filters before injection.
Ethyl-5-phenylbarbituric acid (phenobarbital) was Placebo formulations contained 5-ethyl-5-
obtained from ICN Hungary (Tiszavasvari,´ Hun- phenylbarbituric acid, pyridoxine, phenytoin, mi-
gary), HCl pyridoxine was from F. Hoffmann La crocrystalline cellulose pH 101 and magnesium
Roche (Basel, Switzerland), phenytoin (diphenyl- stearate.
 M. Candela et al. / J. Chromatogr. A 890 (2000) 273 – 280 275

3. Validation of the method samples. The analyte of interest was well separated
from other components present in the samples. The
The procedures and parameters used for the peak purity was, in all cases, more than 99.0%.
validation of the chromatographic method developed
in this study are the ones described in the U.S. 3.2. Linearity
Pharmacopoeia (USP) 24, chapter (1225) [6] and
ICH-Guidelines [7]. Results were processed using The linearity of an analytical method is its ability
the Validation Manager Software (Version 1.3., to produce test results that are directly proportional
Merck, France). to the concentration of analyte in samples within a
given range. For the establishment of linearity, a
3.1. Specificity minimum of five different concentrations should be
used. It is also recommended that a specific range,
The ICH documents define specificity as the normally from 80 to 120% of the test concentration,
ability to assess unequivocally the analyte in the is used for the assay of a finished pharmaceutical
presence of components that may be expected to be product.
present, such as impurities, degradation products and A linear curve fit was obtained from five different
matrix components. Other reputable international concentrations of standard solutions in the range 0.40
authorities (IUPAC, AOAC) have preferred the term to 0.60 mg / ml using three replicate injections. The
selectivity, reserving specificity for those procedures regression line was calculated as y 5 a 1 bx, where x
that are completely selective [6]. was the GABOB concentration (mg / ml) and y was
According to Persson et al. [8], a common and the response (peak area expressed as AU). The
serious mistake is referring to a method as specific calibration curve was obtained using the linear least
when it is only selective. This case is especially true squares regression procedure. Fig. 2 shows the
when dealing with chromatographic methods that are regression line and Fig. 3 a standard chromatogram.
not absolute, but only relative, methods of analysis The RSD (relative standard deviation) values for
[8]. According to the USP terminology and ICH the response factors were in the range 0–5% (1.7%),
guidelines, and to avoid any misunderstanding, we and they were considered adequate for verifying the
decided to use the term specificity when referring to linearity of the regression [9].
this validation parameter. The coefficient of correlation (r) value was close
Demonstration of specificity requires the ability of to unity (0.997), hence, there was a linear relation-
the method to show that the procedure is not ship between the amount of GABOB and the detec-
interfered with by the presence of impurities or tor response.
excipients expected to be in the sample matrix. In
practice, this can be done by spiking the drug 3.3. Accuracy
substance or product with appropriate levels of
impurities / excipients and demonstrating that the Accuracy is the closeness of test results obtained
assay result is unaffected by the presence of these by the method to the true value. Accuracy is often
potential interferences [6]. calculated as percent recovery by the assay of
The specificity of the analytical method in this known, added amounts of analyte to the sample [6].
study was determined by comparing the results from The ICH documents recommend that accuracy
the analysis of samples containing degradation prod- should be assessed using a minimum of nine de-
ucts or placebo ingredients with those obtained from terminations over a minimum of three concentration
the analysis of samples containing only 4-amino-3- levels, covering a specific range (i.e., three con-
hydroxybutyric acid. The analyzed samples were centrations and three replicates for each concen-
GABOB standard, placebo formulation spiked with tration) [7].
GABOB, and a tablet that was stressed at 608C in an Accuracy was determined by spiking known
oven for 18 h. amounts of analyte to a placebo sample across the
Fig. 1 shows the chromatograms of the different specified range of the analytical procedure to obtain
276 M. Candela et al. / J. Chromatogr. A 890 (2000) 273 – 280

Fig. 1. Specificity chromatograms. (a) 4-Amino-3-hydroxybutyric acid standard, (b) placebo formulation and 4-amino-3-hydroxybutyric
acid, (c) a stressed tablet at 608C for 18 h.
 M. Candela et al. / J. Chromatogr. A 890 (2000) 273 – 280 277

Fig. 2. Calibration curve.

0.40, 0.50 and 0.60 mg / ml concentrations (80, 100 of analytical procedure in different laboratories, i.e.,
and 120% of test concentration). Table 1 summarises in a collaborative study. In this case, we have studied
the accuracy results, expressed as percent recovery repeatability and intermediate precision.
and relative standard deviation (RSD). The method
showed excellent recovery, with values that were 3.4.1. Repeatability
close to 100% (100.1%). The repeatability of an analytical method refers to
the use of the procedure within a laboratory over a
3.4. Precision short period of time, carried out by the same analyst
with the same equipment. The ICH documents
The USP defines precision as ‘the degree of recommend that repeatability should be assessed
agreement among individual test results when the using a minimum of nine determinations covering
method is applied repeatedly to multiple samplings the specified range (i.e., three concentrations and
of an homogeneous sample’ [6]. Precision may be three replicates for each concentration) or a mini-
measured as repeatability, reproducibility and inter- mum of six determinations of 100% of the test
mediate precision. Reproducibility refers to the use concentration [7].

Fig. 3. Standard chromatogram (100% of labeled concentration).

278 M. Candela et al. / J. Chromatogr. A 890 (2000) 273 – 280

Table 1
Results of accuracy determination a
Theoretical concentration Experimental concentration Recovery (%)
(mg / ml) (mg / ml)
0.40 0.4029 100.72
0.40 0.4032 100.80
0.40 0.4019 100.47
0.50 0.4977 99.54
0.50 0.4969 99.38
0.50 0.5041 100.82
0.60 0.5958 99.30
0.60 0.5970 99.50
0.60 0.6011 100.18
x̄ 100.1
%R.S.D. 0.4
a
Each number represents individual HPLC measurements.

We considered nine determinations covering the to be used [7]. The most interesting study in our
specified range for our procedure. Standard solutions Quality Control Laboratory is to check the viability
of 0.40, 0.50 and 0.60 mg / ml spiked into placebo of the proposed method using different equipment.
samples were injected (three replicates each) and the The extraction procedure, column, mobile phase and
method was calibrated with three standard solutions the rest of the conditions were the same. The
(0.40, 0.50 and 0.60 mg / ml). The RSD value samples injected into the second HPLC system
(0.68%) indicates that the proposed HPLC method (Perkin-Elmer) were also the same as the ones used
shows acceptable repeatability (Table 2). for the repeatability study. The results obtained by
the two HPLCs were comparable. Table 3 presents
3.4.2. Intermediate precision the individual values obtained for the intermediate
The intermediate precision of an analytical method precision study. The results obtained from both
is the degree of agreement of test results obtained by HPLC systems were similar, although a slight vari-
the analysis of the same sample under various ation was observed at the 120% level. The RSD
conditions (typical variations include different lab- value (1.27%) indicates that the proposed HPLC
oratories, analysts, equipment, etc.) [10]. The extent method shows acceptable intermediate precision.
this parameter should be studied depends on the
circumstances under which the procedure is intended 3.5. Range

Table 2 The range of an analytical method is the interval

Results of repeatability determinations a between the upper and lower levels of analyte
Theoretical concentration Experimental concentration (including these levels) that have been demonstrated
(mg / ml) (mg / ml) to be determined with acceptable precision, accuracy
0.40 0.4029 and linearity using the procedure, as written [6]. The
0.40 0.4032 method developed in our laboratory showed its
0.40 0.4019 suitability for GABOB determinations in the con-
0.50 0.4977 centration range from 0.40 to 0.60 mg / ml.
0.50 0.4969
0.50 0.5041
0.60 0.5958 3.6. Stability of solutions
0.60 0.5970
0.60 0.6011 The stability of solutions was demonstrated by
a
RSD: 0.68%. Each number represents individual HPLC mea- analysing the 100% test concentration (0.50 mg / ml)
surements. and the same spiked into placebo just prepared and
 M. Candela et al. / J. Chromatogr. A 890 (2000) 273 – 280 279

Table 3
Results of intermediate precision determinations a
Theoretical concentration Experimental concentration Experimental concentration
(mg / ml) (mg / ml) HPLC 1 b (mg / ml) HPLC 2 c
0.40 0.4029 0.4013
0.40 0.4032 0.4008
0.40 0.4019 0.3997
0.50 0.4977 0.4945
0.50 0.4969 0.4907
0.50 0.5041 0.4947
0.60 0.5958 0.5837
0.60 0.5970 0.5849
0.60 0.6011 0.5847
a
RSD: 1.7%. Each number represents individual HPLC measurements.
HPLC1: samples analysed using a Merck–Hitachi La Chrom  high-performance liquid chromatograph.
b

c
HPLC2: samples analysed using a Perkin-Elmer high-performance liquid chromatograph.

after 6 days of refrigerated storage. The solutions did significant change in the system’s suitability parame-
not undergo any decomposition. The results of the ters (retention time, capacity factor k9, asymmetry
system suitability test and area values did not show and number of theoretical plates). The results are
significant differences. shown in Tables 4 and 5. The values presented are
the average of two different measurements.
3.7. Robustness

The robustness of an analytical procedure is a 4. Conclusion

measure of its capacity to remain unaffected by
small, but deliberate, variations in method parame- We developed a novel and straightforward ana-
ters, and provides an indication of its reliability lytical method for the determination of 4-amino-3-
during normal usage. Different types of designs are hydroxybutyric acid concentrations in pharmaceu-
used in robustness testing, and ICH guidelines tical tablets. The main advantage of this HPLC
recommend its evaluation during the development procedure over the previous ones described for the
phase of a method, and not at the end, as occurred in same compound is that it does not require deri-
this study [11]. vatization.
We analysed the influence of different pH values We studied the validation parameters according to
and mobile phase counter-ion concentrations. pH USP and ICH recommendations and demonstrated
variations of the mobile phase produced important that the proposed new method is specific, linear,
changes in the retention time and peak shape, while accurate and precise, within the established range.
the counter-ion concentration did not show any Therefore, the HPLC procedure developed was

Table 4
Results of robustness (effect of counter-ion concentration in the mobile phase) study a
Samples Counter-ion Retention k9(capacity Asymmetry N (number of Area
concentration time (min) factor) theoretical plates)
GABOB 0.009 M 16.71 10.94 1.54 7346 224 240
standard 0.01 M 17.26 12.28 1.79 8049 222 660
0.011 M 16.29 10.64 1.55 7490 218 448
Placebo 0.009 M 17.27 11.70 1.57 7444 22 488
1 0.01 M 17.22 11.30 1.72 8001 224 745
GABOB 0.011 M 16.41 10.73 1.58 7615 219 450
a
Each value represents the average of two injections.
280 M. Candela et al. / J. Chromatogr. A 890 (2000) 273 – 280

Table 5
Results of robustness (effect of mobile phase pH) study a
Samples pH Retention k9 (capacity Asymmetry N (number of Area
time (min) factor) theoretical plates)
GABOB 2.0 13.29 8.48 1.20 6780 212 138
standard 2.4 17.26 12.28 1.79 8049 222 660
3.0 19.28 12.77 0.89 5762 171 100
Placebo 2.0 13.08 8.33 1.10 6389 215 665
1 2.4 17.22 11.30 1.72 8001 224 745
GABOB 3.0 19.29 12.78 0.85 5249 164 748
a
Each value represents the average of two injections.

proved to be suitable for the determination of ˜

[4] G. Bugueno, I. Escriche, J.A. Serra, O. Munoz, J. Rastrepo,
Alimentaria 299 (1999) 77.
GABOB concentrations in pharmaceutical finished
[5] W. Zhang, X. Sun, Q. She, X. Zhang, Sepu 16 (6) (1999)
products. 539.
[6] U.S. Pharmacopoeia 24 / National Formulary 19, United
States Pharmacopeial Convention, Rockville, MD, 1995, p.
Acknowledgements 2149, section 1225.
[7] ICH-Guidelines. Validation of Analytical Procedures: Meth-
odology (CPMP/ ICH / 281 / 95, Final) (ICH Topic Q2B, Step
The authors thank Dra. M. Idoya Gastearena for 4).
her contribution. [8] B.A. Persson, J. Vessman, R.D. McDowall, LC?GC 15 (10)
(1997) 944.
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[9] AEFI, Validacion ´ ´
analıticos, ´ de Nor-
Comision
mas de Buena Fabricacion´ y Control de Calidad. Asociacion ´
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