OPTIMIZATION OF LIQUID CHROMATOGRAPHIC METHOD FOR THE SEPARATION OF FOLIC ACID AND ITS TWO IMPURITIES

This article was downloaded by: [University of South Carolina ]On: 21 April 2013, At: 05:10Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Instrumentation Science & TechnologyPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/list20

OPTIMIZATION OF LIQUIDCHROMATOGRAPHIC METHOD FOR THESEPARATION OF FOLIC ACID AND ITS TWOIMPURITIESAnđelija Malenović a , Marko Jovanović a , Sonja Petrović a , NađaKostić a , Ana Vemić a & Biljana Jančić-Stojanović aa Faculty of Pharmacy, Department of Drug Analysis, University ofBelgrade, Belgrade, SerbiaAccepted author version posted online: 19 Jan 2012.Version ofrecord first published: 01 May 2012.

To cite this article: Anđelija Malenović , Marko Jovanović , Sonja Petrović , Nađa Kostić , Ana Vemić& Biljana Jančić-Stojanović (2012): OPTIMIZATION OF LIQUID CHROMATOGRAPHIC METHOD FOR THESEPARATION OF FOLIC ACID AND ITS TWO IMPURITIES, Instrumentation Science & Technology, 40:2-3,138-149

To link to this article: http://dx.doi.org/10.1080/10739149.2011.651670

PLEASE SCROLL DOWN FOR ARTICLE

Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions

This article may be used for research, teaching, and private study purposes. Anysubstantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,systematic supply, or distribution in any form to anyone is expressly forbidden.

The publisher does not give any warranty express or implied or make any representationthat the contents will be complete or accurate or up to date. The accuracy of anyinstructions, formulae, and drug doses should be independently verified with primarysources. The publisher shall not be liable for any loss, actions, claims, proceedings,demand, or costs or damages whatsoever or howsoever caused arising directly orindirectly in connection with or arising out of the use of this material.

http://www.tandfonline.com/loi/list20

http://dx.doi.org/10.1080/10739149.2011.651670

http://www.tandfonline.com/page/terms-and-conditions

Instrumentation Science and Technology, 40:138–149, 2012Copyright © Taylor & Francis Group, LLCISSN: 1073-9149 print/1525-6030 online DOI: 10.1080/10739149.2011.651670

OPTIMIZATION OF LIQUID CHROMATOGRAPHIC METHOD FOR THE SEPARATION OF FOLIC ACID AND ITS TWO IMPURITIES

Andelija Malenovic, Marko Jovanovic, Sonja Petrovic, Nada Kostic, Ana Vemic, and Biljana Jancic-StojanovicFaculty of Pharmacy, Department of Drug Analysis, University of Belgrade, Belgrade, Serbia

This paper was submitted as part of a special issue entitled “Instrumentation and Stationary Phases for Liquid Chromatography” organized by Dr. Constantinos Zacharis of the Aristotle University of Thessaloniki.

Address correspondence to Biljana Jancic -Stojanovic , Faculty of Pharmacy, Department of Drug Analysis, University of Belgrade, Vojvode Stepe 450, Belgrade, Serbia. E-mail: jancic.s [email protected]

In this article, the multiobjective optimization of reversed-phase high-performance liquid chromatography (RP-HPLC) method for the analysis of folic acid and its two impurities, p-aminobenzoic acid and N-(4-aminobenzoyl)-L-glutamic acid, is presented. During the preliminary study, the independent variables whose impact should be further examined in the optimization were defined (acetonitrile content in the mobile phase, molarity of sodium 1-heptanesulfonate in the aqueous phase, and pH of the aqueous phase). The face-centered central composite design was chosen for the method optimization. As the dependent variables, the separation between the impurities and the retention factor of folic acid were followed. The analysis of variance (ANOVA) test was done, and good correlation among results was confirmed. For the more accurate and reliable optimization, Derringer’s desirability function was applied. On the basis of restrictions adopted for the selected targets, the resulting optimal composition of the mobile phase was acetonitrile –3 mM sodium 1-heptanesulfonate (5.3:94.7 V/V), pH of the aqueous phase adjusted to 2.1 with the concentrated ortho-phosphoric acid. Furthermore, the method was subjected to method validation. It proved to be reliable and suitable for the routine analysis of investigated substances in active pharmaceutical ingredients, as well as in pharmaceutical preparations.

Keywords p-aminobenzoic acid, N-(4-aminobenzoyl)-L-glutamic acid, central c om posite design, desirability-based optimization, folic acidD

ownl

oade

d by

[U

nive

rsity

of

Sout

h C

arol

ina

] at

05:

10 2

1 A

pril

2013

Optimization of Liquid Chromatographic Method 139

INTRODUCTION

In modern chromatographic analysis, optimization of a separation is principally directed by the following goals: (1) to separate better for higher resolution, (2) to separate faster for shorter retention time, (3) to see more, resulting in a lower detection limit, (4) to separate at lower cost, or (5) to separate more, resulting in higher throughput.[1] Mainly, higher resolution and shorter retention time are the issues of interest. For this purpose, several approaches are available, e.g., experimental design, multi-objective optimization, artificial neural networks, computer-assisted procedures, etc. In this article, the desirability-based approach was selected for the optimization of ion pair liquid chromatographic method for the analysis of folic acid and its two impurities, p-aminobenzoic acid (PABA) and N-(4-aminobenzoyl)-L-glutamic acid (LG).

For the determination of folic acid, PABA, and LG, British Pharmacopoeia gives two separate liquid chromatography (LC) procedures.[2] A literature survey shows an article dealing with LC determination of folic acid in the presence of PABA and LG,[3] as well as one that presents densitometric determination of LG in preparations of folic acid.[4] On the other hand, for the analysis of folic acid in multivitamin tablets, liquid chromatographic methods combined with mass spectrometry or tandem mass spectrometry are described.[5–8] For the analysis of multivitamin praparations, several high-performance liquid chromatography (HPLC) methods using UV,[9,10] diode array detector,[11] or diode array detector-fluorescence detector/mass spectrometric detection[12] were described. Determination of folic acid by capillary electrophoresis with chemiluminescence detection[13] and by microemulsion electrokinetic chromatography were found.[14]

The analysis of folic acid in the presence of these two particular impurities is important, as they are the products of hydrolytic degradation of folic acid,[2] and as such should be evaluated during the shelf-life of the corresponding pharmaceutical preparation. In a previously published article,[3] the authors have applied the official United States Pharmacopoeia (USP) assay method for folic acid in bulk drug on the determination of impurities in the pharmaceutical preparations containing folic acid. The major problem is that with the proposed method, adequate selectivity was not achieved, i.e., PABA and LG were not separated properly from the other tablets’ components. In addition, as the authors applied method for a purpose it was not intended for, it had to be properly validated. It could not be validated, and therefore method’s applicability in routine analysis is not possible and reliable. For that reason, in order to overcome the lack of an already published method, in this study a new LC method for the simultaneous determination of folic acid, PABA, and LG was developed. Furthermore, the advancement of method optimization step was achieved

Dow

nloa

ded

by [

Uni

vers

ity o

f So

uth

Car

olin

a ]

at 0

5:10

21

Apr

il 20

13

140 A. Malenovic et al.

by the application of Derringer’s desirability function to simultaneously optimize the two selected responses (separation between PABA and LG and retention factor of folic acid). Finally, in order to confirm the adequacy for routine application, the proposed method was validated with determination of selectivity, linearity, accuracy, precision, limit of detection, and limit of quantification determined.

EXPERIMENTAL

Chemicals

Reference substances of folic acid (Supelco Analytical, lot LB58887, purity 98.0%), PABA (Merck, lot K26714002929, purity 99.5%), and LG (Sigma, lot 028K1137, purity 98.0%) were used to prepare standard solutions. All reagents used were of an analytical grade. Acetonitrile – HPLC gradient grade (Sigma, St. Louis, MO, USA), sodium 1-heptanesulfonate monohydrate obtained from Fluka, and water – HPLC grade were used to prepare a mobile phase. Ortho-phosphoric acid (J. T. Baker, Deventer, Netherlands) was used to adjust the pH of the mobile phase. Tablets containing 5 mg of folic acid and, according to the manufacturer’s shelf life specification, not more than 0.5% of PABA and 2% of LG were used for the preparation of the solutions for precision testing.

Chromatographic Conditions

The chromatographic system Finnigan Surveyor Thermo Scientific consisted of Finnigan Surveyor LC Pump Plus, Finnigan Surveyor UV/Vis Plus Detector, Finnigan Surveyor Autosampler Plus, and ChromQuest for data collection. The samples were introduced through a partial loop in 5 mL volume. For the separation the XBridge C8 3.0 × 100 mm, 3.5 µm particle size column was used. UV detection was performed at 269 nm. Mobile phases were prepared according to the plan of experiments given in Table 1. The resulting mobile phases were filtered through a 0.45 µm membranes filter (Alltech, Loceren, Belgium). The flow rate was 1 µL min–1, and the column temperature was 30 °C.

Software

Experimental design, data analysis, and desirability function calculations were performed by using Design-Expert® 7.0.0 (Stat-Ease Inc., Minneapolis, MN).

Dow

nloa

ded

by [

Uni

vers

ity o

f So

uth

Car

olin

a ]

at 0

5:10

21

Apr

il 20

13


Standards and Solutions

Solvent PreparationThe solvent was prepared by mixing 50 mL of acetonitrile and 950 mL

of 3 mm sodium 1-heptanesulfonate.

Standard Solutions for Method OptimizationStock solutions were prepared by dissolving folic acid in 0.1 M NaOH,

and then for the further dilution up to the concentration of 100 mg mL–1, the solvent was used. Stock solutions for impurities were prepared in the solvent to obtain concentrations of 10 mg mL–1 for both of them.

Solutions for the Selectivity EstimationTo prove the selectivity of the proposed method, a placebo mixture

was prepared. The mixture was treated in the same manner as tablet mass used for the preparation of sample solution. A laboratory standard mixture, as well as the sample solution containing 100 µg mL–1 of folic acid, 0.40 µg mL–1 of PABA, and 1.5 µg mL–1 LG, was used for selectivity proving.

TABLE 1 Plan of Experiment and the Obtained Results

No. A B C α imp1/imp2 kF

1 5 (–1)* 3 (–1) 2 (–1) 1.410 25.561 2 7 (+1) 3 (–1) 2 (–1) 1.117 8.975 3 5 (–1) 5 (+1) 2 (–1) 1.448 34.674 4 7 (+1) 5 (+1) 2 (–1) 1.165 11.915 5 5 (–1) 3 (–1) 2.5 (+1) 1.320 22.906 6 7 (+1) 3 (–1) 2.5 (+1) 1.051 7.993 7 5 (–1) 5 (+1) 2.5 (+1) 1.408 28.428 8 7 (+1) 5 (+1) 2.5 (+1) 1.120 9.838 9 5 (–1) 4 (0) 2.25 (0) 1.402 27.30210 7 (+1) 4 (0) 2.25 (0) 1.112 10.54511 6 (0) 3 (–1) 2.25 (0) 1.197 13.65612 6 (0) 5 (+1) 2.25 (0) 1.247 16.79413 6 (0) 4 (0) 2 (–1) 1.221 15.50914 6 (0) 4 (0) 2.5 (+1) 1.153 12.73715 6 (0) 4 (0) 2.25 (0) 1.240 15.69516 6 (0) 4 (0) 2.25 (0) 1.257 16.37717 6 (0) 4 (0) 2.25 (0) 1.234 15.40418 6 (0) 4 (0) 2.25 (0) 1.240 15.825

A – content of acetonitrile (%); B – content of sodium 1-heptanesulfonate (mmol L–1); C – pH of the water phase.

α imp1/imp2 – separation factor between p-aminobenzoic acid and N-(4-aminobenzoyl)-L-glutamic acid, respectively. kF – retention factor for folic acid. *in the brackets coded values for factors levels are given.

Dow

nloa

ded

by [

Uni

vers

ity o

f So

uth

Car

olin

a ]

at 0

5:10

21

Apr

il 20

13


Solutions for the Linearity EstimationFor the calibration curve, six solutions containing folic acid in the

concentration range from 50 µg mL–1 to 150 µg mL–1 were prepared in the solvent. Six solutions of impurities were prepared in the solvent in the concentration range from 0.1 µg mL–1 to 0.6 µg mL–1 for PABA and from 0.5 µg mL–1 to 2.5 µg mL–1 for LG.

Solutions for the Precision EstimationTo check the precision of the proposed method, three series of sample

solution were prepared with three solutions for each sample concentration. The standard solution containing 0.4 mg mL–1 of PABA and 1.5 mg mL–1 of LG (solution iP) was prepared in the solvent. The quantity of the pulverized tablet mass corresponding to 100 mg of folic acid was placed into a 100 mL volumetric flask, than spiked with 1 mL of solution iP, and finally 70 mL of 0.1 M NaOH was added. The extraction was done by sonication for 15 min. After sonication, the volumetric flask was filled to the mark, and the resulting suspension was filtered using Whatman filter paper. Further dilutions were prepared using the solvent in order to obtain the following concentrations:

1. Solution 1 containing 80 µg mL–1 of folic acid, 0.32 µg mL–1 of PABA, and 1.2 µg mL–1 LG



Solutions for the Accuracy EstimationFor the accuracy testing, the laboratory mixture containing a placebo

as well as folic acid, PABA, and LG was prepared in solvent in the ratio corresponding to the investigated tablets. The laboratory mixture was treated in the same manner as tablet mass used for the preparation of solutions for estimating the precision. Then the three series of dilutions calculated as 80%, 100%, and 120% at concentrations corresponding to those in tablets, with three solutions for each concentration mixture, were prepared:

1. Mixture 1 containing 80 µg mL–1 of folic acid, 0.32 µg mL–1 of PABA, and 1.2 µg mL–1 LG

2. Mixture 2 containing 100 µg mL–1 of folic acid, 0.40 µg mL–1 of PABA, and 1.5 µg mL–1 LG

3. Mixture 3 containing 120 µg mL–1 of folic acid, 0.48 µg mL–1 of PABA, and 1.8 µg mL–1 LG.

Dow

nloa

ded

by [

Uni

vers

ity o

f So

uth

Car

olin

a ]

at 0

5:10

21

Apr

il 20

13


RESULTS AND DISCUSSION

Establishing the appropriate chromatographic conditions for the identification and quantification of impurities could be difficult due to (a) the small quantities present in the investigated sample; (b) structural similarities between impurities and the active pharmaceutical ingredient from which they originate; and (c) quite different polarities of the impurities, mostly those emerging after the hydrolytic degradation of pharmaceutical substance. During the preliminary investigation, the impact of chromatographic conditions on the separation of analyzed substances was investigated in order to elucidate the factors that affect the system the most. For that reason, the analyses were performed on C8 and C18 columns with varying content of methanol or acetonitrile and different types of water phases. Namely, different concentrations of di-potassium-hydrogenphosphate and sodium 1-heptanesulfonate in water phase were tested. The influence of the pH of the water phase was also investigated. The factors, whose impact should be further examined in the optimization, acetonitrile content in the mobile phase, concentration of sodium 1-heptanesulfonate in the aqueous phase, and pH of the aqueous phase, were selected. All other factors (column, flow rate, and detection wavelength) were kept on defined levels.

The next step was the selection of the appropriate experimental design. The most adequate designs for the optimization purpose are second order symmetrical designs such as CCD. In this study, a face-centered charge-coupled device (CCD) with 23 full factorial design, ±1 star design, and four replications in central point was used for the definition of the plan of experiments. The chosen factors and their levels are presented in Table 1. As the system outputs, the separation between PABA and LG (αimp1/imp2) and retention factor of folic acid (kF) were followed. These outputs were selected, as they can provide the most realistic representation of the analyzed system. Namely, when the impurities were separated adequately, folic acid retention time was inappropriately long. Obviously, the optimization shoud be directed towards the αimp1/imp2 maximization with the reasonable run time (kF as low as reasonable possible). The next step was the realization of the experiments according to the defined plan, and the resulting responses are given in Table 1. The appropriate calculations were done via Design-Expert 7.0.0.

The obtained mathematical models, which represent the relationship between dependent and independent variables, were defined. For both responses, the quadratic response model was suggested as the most adequate. The calculated coefficients of response model for coded factor levels are given in Table 2.

Dow

nloa

ded

by [

Uni

vers

ity o

f So

uth

Car

olin

a ]

at 0

5:10

21

Apr

il 20

13


The adequacy of the proposed model was eveluated using the analysis of variance (ANOVA) test, confirming that it can be used as the appropriate basis for further optimization. For both models, p-values were < 0.05, implying that these models are significant. The “response to deviation ratio” is used as a measure of the adequate response, and a ratio greater than 4 is desirable.[15] In this study, the ratio was found to be 30.166 and 42.609 for αimp1/imp2 and kF models, respectively. As a measure of reproducibility of the model, the % CV is adopted, and generally a model can be considered reasonably reproducible if this value is less than 10%.[15] The CV for both models was found to be less than 10% (1.45% and 4.64% for αimp1/imp2 and kF models, respectively). Also, in the present study, the adjusted R2 values for both response models were well within the acceptable limits of R2 ³ 0.80,[16] which confirmed that the experimental data were fitted well with the second-order polynomial equations (Table 2).

The predicted models can also be presented as a perturbation plots in order to gain a better understanding of the results [Figures 1a (αimp1/

imp2) and 1b (kF)].[17] In case of the optimization design, this graph helps

to compare the effect of all the factors at a particular point in the design space. The response is plotted by changing only one factor over its range while keeping the other factors at a constant level. In this investigation, the reference point was set at the midpoint (coded 0) of all the factors. The response changes as each factor moves from a chosen reference point, while all other factors remain constant at the reference value. A steep

TABLE 2 Coefficients of Quadric Modely = b0 + b1A + b2B + b3C + b12AB + b13AC + b23AD + b11A

2 + b22B2 + b33C

2

Selectivity Factor (αimp1/imp2)* Retention Factor of Folic Acid (kF)

Coefficient p-value Coefficient p-valuebo 1.23 <0.0001** 15.41 <0.0001**b1 –0.14 <0.0001** –8.96 <0.0001**b2 0.029 0.0009** 2.26 <0.0001**b3 –0.031 0.0006** –1.47 0.0004**b12 –0.0011 0.8642 –1.23 0.0024**b13 0.0024 0.7189 0.73 0.0324**b23 0.0089 0.2009 –0.59 0.0719b11 0.043 0.0046** 3.92 <0.0001**b22 0.0076 0.5079 0.22 0.6559b33 –0.027 0.0366** –0.88 0.1083R2 0.9887 0.9947Adj. R2 0.9761 0.9887

*imp1 – p-aminobenzoic acid; imp2 – N-(4-aminobenzoyl)-L-glutamic acid. **Coefficients significant for p-value

Dow

nloa

ded

by [

Uni

vers

ity o

f So

uth

Car

olin

a ]

at 0

5:10

21

Apr

il 20

13


slope or curvature in a factor indicates that the response is sensitive to that factor. On the other hand, a relatively flat line shows insensitivity to change in that particular factor. From the given plot, it can be seen that factor A (acetonitrile content) mostly affected both responses. Although the influences of factor B (content of sodium 1-heptansulfonate) and factor C (pH of the water phase) are quite similar in intensity, they have the opposite effect, i.e., for factor B, the increase is decreasing the response while for C the increase is increasing the response (Figure 1).

Furthermore, according to the defined aim, two responses with different targets were optimized by Derringer’s desirability. This function is defined as the geometric mean, sometimes weighted, of the individual desirability functions.[18] Equation (1) defines Derringer’s desirability function:

∏ i

nrni

i=1

D= d (1)

where D is the global desirability function value, di is the partial desirability function value calculated for each of the n responses, and ri is the relative importance assigned to the each of the responses i. Relative importance ri is a comparative scale used for weighting each of the resulting di, and it can be varied from the least important (ri = 1) to the most important (ri = 5). The other user-specified parameter that can be used to stress the defined goals is weight. Weights lower than 1 give less emphasis to the goal, while weights greater than 1 give more emphasis to the goal. In both cases, di varies in a nonlinear way while approaching the desired value, while with a weight of 1 di varies in a linear way.[18] A value of D close to 1 implies that the combination of the different criteria is globally optimal, so that the

FIGURE 1 Perturbation plots for (a) αimp1/imp2 and (b) kF.

Dow

nloa

ded

by [

Uni

vers

ity o

f So

uth

Car

olin

a ]

at 0

5:10

21

Apr

il 20

13


response values are near defined target values. In the present report, the goal was to maximize the response for αimp1/imp2 in the range from 1.25 to 1.35. The weight was set to 1. For the kF, the goal was minimization in the range from 22 to 25, while the weight was adjusted to 1. The importance was set to 3 for both responses.

Derringer and Suich[19] proposed the adequate desirability functions depending on whether a particular response is to be maximized, minimized, or reach the desired target value.

On the basis of adopted conditions and restrictions given above, the optimal composition of the mobile phase was predicted as follows: acetonitrile –3mM sodium 1-heptanesulfonate (5.3:94.7 V/V); the pH of the aqueous phase adjusted to 2.1 with the concentrated ortho-phosphoric acid. The corresponding global desirability was 1.000. The predicted response values (for αimp1/imp2 was 1.35 and for was kF 21.92) were confirmed experimentally. The observed difference between the predicted and experimental responses is found to be in good agreement and well within the acceptance limits.

As the methods used in pharmaceutical analysis must be sufficiently accurate, selective, sensitive, and precise to conform to the regulatory requirements as set out in the relevant guideline ICH Q2(R1)—Validation of Analytical Procedures: Text and Methodology,[20] the optimized method was validated and the relevant parameters were tested. In order to prove the selectivity of the established method, the representative chromatogram of folic acid and analyzed impurities laboratory mixture were compared to the placebo mixture chromatogram and sample chromatogram (Figure 2). The suggested method proved to be selective, since no other peaks were observed at the retention times of the examined substances.

The next step was robustness testing, which was done by the “one factor at a time” method. Namely, one of the investigated factors (acetonitrile content in the mobile phase, concentration of sodium 1-heptanesulfonate in the aqueous phase, and pH of the aqueous phase) was varied while the others were kept at a constant level. From the conducted investigations, we concluded that the pH of the aqueous phase followed by the content of acetonitrile in the mobile phase have significant influence on the resolution of PABA and LG, as well as on the retention behavior of folic acid. Accordingly, those factors must be strictly controlled, while the content of sodium 1-heptanesulfonate in the aqueous phase could be increased up to 3.5 mm with no effect on the investigated substances.

Furthermore, limit of detection (LOD) and limit of quantification (LOQ) as important parameters for the quantitative analysis were experimentally determined based on signal-to-noise ratio of 3:1 and 10:1 for LOQ and LOD, respectively. For PABA, the determined LOD value was 0.05 µg mL–1 and LOQ was 0.1 µg mL–1, while for LG the defined LOD was 0.2 µg mL–1 and LOQ was 0.5 µg mL–1.

Dow

nloa

ded

by [

Uni

vers

ity o

f So

uth

Car

olin

a ]

at 0

5:10

21

Apr

il 20

13


Linear relationships of the peak area over the mentioned concentration range for folic acid, PABA, and LG were obtained. All linearity parameter values are within the linearity acceptance criteria. The calculated regression parameters are given in Table 3.

For estimating another important validation parameter, method precision, the values for relative standard deviation (RSD) were determined. Fulfillment of the required criterion indicates that the proposed LC method is precise. RSD values for folic acid were from 0.3% to 1.7%, from 1.1% to 3.1% for PABA, and from 0.8% to 2.4% for LG. All the obtained results are in a good agreement with defined

FIGURE 2 (a) Placebo mixture chromatogram. (b) The representative chromatogram of standard solution mixture containing 100 µg mL–1 of folic acid, 0.40 µg mL–1 of PABA, and 1.5 µg mL–1 LG: tr = min (PABA), tr = min (LG), and tr = min (folic acid) [mobile phase: acetonitrile – 3mM sodium 1-heptanesulfonate (5.3:94.7 V/V), pH of the aqueous phase adjusted to 2.1 with the concentrated ortho-phosphoric acid; flow rate: 1 mL min–1; λ = 269 nm; column temperature: 30 °C]. (c) The chromatogram of sample solution containing 100 µg mL–1 of folic acid.

Dow

nloa

ded

by [

Uni

vers

ity o

f So

uth

Car

olin

a ]

at 0

5:10

21

Apr

il 20

13


requirements.[21] The content of the folic acid in the analyzed tablets was 92.70% with 0.40% of PABA and 0.78% of LG.

To evaluate the method accuracy, recovery values for laboratory mixtures were calculated, and all of them were within the acceptable criteria,[21] as the values for folic acid were from 102.2% to 102.5%, from 78.3% to 81.5% for PABA, and from 94.4% to 102.0% for LG.

CONCLUSION

Desirability-based optimization was used for the multiobjective optimization of liquid chromatographic separation of folic acid, p-aminobenzoic acid, and N-(4-aminobenzoyl)-L-glutamic acid. A central composite design was applied to define the plan of experiments in order to examine the two selected outputs. The predicted models were also presented as a perturbation plots for αimp1/imp2 and kF, which facilitated understanding of the obtained results. For the definition of the global optimal conditions, targets for selected outputs were defined, and weights, importance coefficients, and ranges were set. With respect to the selected conditions and restrictions, the optimization procedure was carried out, and the optimal mobile phase composition was predicted. Such an approach to method optimization proved to be valuable in optimizing parameters controlling separation in the proposed LC method. Furthermore, the method was subjected to validation. It proved to be reliable and suitable for the routine analysis of folic acid, p-aminobenzoic acid, and N-(4-aminobenzoyl)-L-glutamic acid in active pharmaceutical ingredients, as well as in pharmaceutical preparations.

ACKNOWLEDGMENTS

The authors thank the Ministry of Science and Technological Development of the Republic of Serbia for supporting these investigations in Project 172052.

TABLE 3 Linearity

Parameter Folic acid p-Aminobenzoic acidN-(4-Aminobenzoyl)-L-

glutamic acid

Range (µg mL–1) 50–150 0.1–0.6 0.5–2.5Slope 56.48 56.74 19.85Intercept 106.81 –1.46 3.15Coefficient of correlation

0.9998 0.9995 0.9999

Dow

nloa

ded

by [

Uni

vers

ity o

f So

uth

Car

olin

a ]

at 0

5:10

21

Apr

il 20

13


REFERENCES

1. S. Kromidas, Ed. HPLC Made to Measure: A Practical Handbook for Optimization; Wiley-VCH: Weinheim, Germany, 2006.

2. The British Pharmacopoeia; The British Pharmacopoeia Commission Secretariat of the Medicines and Healthcare Products Regulatory Agency, London, UK, 2007.

3. Kłaczkow, G.; Anuszewska, E. L. Determination of impurities in pharmaceutical preparations containing folic acid. Acta Pol. Pharm. 2006, 63, 391–394.

4. Krzek, J.; Kwiecien, A. Densitometric determination of impurities in drugs, Part IV. Determination of N-(4-aminobenzoyl)-L-glutamic acid in preparations of folic acid. J. Pharm. Biomed. Anal. 1999, 21, 451–457.

5. Goldschmidt, R. J.; Wolf, W. R. Simultaneous determination of water-soluble vitamins in SRM 1849 infant/adult nutritional formula powder by liquid chromatography–isotope dilution mass spectrometry. Anal. Bioanal. Chem. 2010, 397, 471–481.

6. Chen, Z.; Chen, B.; Yao, S. High-performance liquid chromatography/electrospray ionization-mass spectrometry for simultaneous determination of taurine and 10 water-soluble vitamins in multivitamin tablets. Anal. Chim. Acta 2006, 569, 169–175.

7. Chen, P.; Wolf, W. R. LC/UV/MS-MRM for the simultaneous determination of water-soluble vitamins in multi-vitamin dietary supplements. Anal. Bioanal. Chem. 2007, 387, 2441–2448.

8. Nelson, B. C.; Sharpless, K.; Sander, L. C. Quantitative determination of folic acid in multivitamin/multielement tablets using liquid chromatography/tandem mass spectrometry. J. Chromatogr. A 2006, 1135, 203–211.

9. Höller, U.; Brodhag, C.; Knöbel, A.; Hofmann, P.; Spitzer, V. Automated determination of selected water-soluble vitamins in tablets using a bench-top robotic system coupled to reversedphase (RP-18) HPLC with UV detection. J. Pharm. Biomed. Anal. 2003, 31, 151–158.

10. Amidžic, R.; Brboric, J.; Cudina, O.; Vladimirov, S. RP-HPLC determination of vitamins B1, B3, B6, folic acid and B12 in multivitamin tablets. J. Serb. Chem. Soc. 2005, 70, 1229–1235.

11. Li, H. B.; Chen, F. Simultaneous determination of nine water-soluble vitamins in pharmaceutical preparations by high-performance liquid chromatography with diode array detection. J. Sep. Sci. 2001, 24, 271–274.

12. Chen, P.; Atkinson, R.; Wolf, W. R. Single-laboratory validation of a high-performance liquid chromatographic-diode array detector-fluorescence detector/mass spectrometric method for simultaneous determination of water-soluble vitamins in multivitamin dietary tablets. J. AOAC Int. 2009, 92, 680–687.

13. Zhaoa, S.; Yuan, H.; Xie, C.; Xiao, D. Determination of folic acid by capillary electrophoresis with chemiluminescence detection. J. Chromatogr. A 2006, 1107, 290–293.

14. Aurora-Prado, M. S.; Silva, C. A.; Tavares, M. F. M.; Altria, K. D. Determination of folic acid in tablets by microemulsion electrokinetic chromatography. J. Chromatogr. A 2004, 1051, 291–296.

15. Beg, Q.; Sahai, V.; Gupta, R. Statistical media optimization and alkaline protease production from Bacillus mojavensis in a bioreactor. Process Biochem. 2003, 39, 203–209.

16. Lundstedt, T.; Seifert, E.; Abramo, L.; Thelin, B.; Nystrom, A.; Pettersen, J.; Bergman, R. Experimental design and optimization. Chemom. Intell. Lab. Syst. 1998, 42, 3–40.

17. Decaestecker, T. N.; Lambert, W. E.; Van Peteghem, C. H.; Deforce, D.; Van Bocxlaer, J. F. Optimization of solid-phase extraction for a liquid chromatographic-tandem mass spectrometric general unknown screening procedure by means of computational techniques. J. Chromatogr. A 2004, 1056, 57–65.

18. Sivakumar, T.; Manavalan, R.; Muralidharan, C.; Valliappan, K. An improved HPLC method with the aid of a chemometric protocol: Simultaneous analysis of amlodipine and atorvastatin in pharmaceutical formulations. J. Sep. Sci. 2007, 30, 3143–3153.

19. Derringer, G.; Suich, R. Simultaneous optimization of several response variables. J. Quality Technol. 1980, 12, 214–219.

20. International Conference on Harmonization of Technical Requirements for Registraton of Pharmaceuticals for Human Use. Guideline Q2(R1) Validation of Analytical Procedures: Text and Methodology. Available at: http://www.ich.org (access date May 2011).

21. Crowther, J. B. Validation of pharmaceutical test methods. In Handbook of Modern Pharmaceutical Analysis; S. Ahuja, S. Scypinski (Eds.); Academic Press: New York, 2001, pp. 415–443.

Dow

nloa

ded

by [

Uni

vers

ity o

f So

uth

Car

olin

a ]

at 0

5:10

21

Apr

il 20

13

Documents

OPTIMIZATION OF LIQUID CHROMATOGRAPHIC METHOD FOR THE SEPARATION OF FOLIC ACID AND ITS TWO IMPURITIES