An Experimental Design Approach to Selecting the Optimum Liquid Chromatographic Conditions for the Determination of Vitamins B1, B2-Phosphate,

B3, B6 and C in Effervescent Tablets Containing Saccharin and Sunset Yellow FCF
C. Nsengiyumva 1. / J. O. D e B e e r 2 / W. Van de Wauw 2 / A. J. Vlietinck 1 / E P a r m e n t i e r 2 1Universitaire Instelling Antwerpen (UIA), Universiteitsplein 1, 2610 Antwerpen (Wilrijk), Belgium 2Instituut voor Hygiene en Epidemiologie (IHE), J. Wytsmanstraat 14, 1050 Brussels, Belgium

Key Words
Column liquid chromatography Vitamin B and C in tablets Experimental design Optimization of separation Multiple regression modeling.

Summary
The ion-pair liquid-chromatographic separation of the water-soluble vitamins thiamine mononitrate, riboflavin phosphate, nicotinamide, pyridoxine hydrochloride, ascorbic acid, saccharin, and the dye Sunset Yellow FCF (El10) has been examined for the analysis of the compounds in effervescent tablets. A Draper-Lin small composite design was used to study the impact on the compounds' retention times and peak-widths (the response variables) of four different mobile phase variables: percentage of methanol as organic modifier, the concentrations of hexanesulfonate as ion pairing reagent and of triethanolamine as competitive base, and pH. The interactions of these variables were also studied. The proposed design enabled derivation of multiple linear regression equations to model each compound's retention time and peak-width at half-height. The statistical reliability of the regression models was established by comparing predicted and experimental values. By introducing the regression models into a spreadsheet program (Excel 5.0), retention times and peak-widths for each compound were calculated at fixed mobile phase pH. The values of all the other combinations of the three mobile phase variables were changed in increments of two units within their examined boundaries, resulting in 225 different rows. For each combination the compounds' calculated retention times and their corresponding peak-widths were sorted in increasing order and the resolution between successive peaks was calcuOriginal 0009-5893/97/06 634-11 $ 03.00/0

lated. The minimum effective resolution (Rs min) between each pair of peaks and the maximum retention time (tR max) in each row were then selected and used to construct contour plots indicating the location of zones of mobile phase parameter combinations where RS min > 1.5 and the analysis was rugged, and where tR max values were minimum. Their common regions resulted in optimum chromatographic separations. Examples are shown of chromatographic separations obtained using mobile phase combinations which were the best compromise of the three criteria selected.

Introduction
Vitamins are vital substances for normal growth, development and health. Their determination is of interest in biochemistry, pharmaceuticals and the food sciences. The water-soluble vitamins (WSVs) including thiamine mononitrate (B1), riboflavin phosphate (B2), nicotinamide (B3), pyridoxine hydrochloride (B6), ascorbic acid (C) and excipients such as saccharin and sunset yellow FCF (El10) that are common ingredients in multivitamin formulations, such as supplementary nutrients, are investigated in this work. Problems of peak coelution, poor peak shapes (thiamine, pyridoxine) and interference from excipient materials, among them El10, and sweeteners such as saccharin are often encountered in simultaneous determination of WSVs by liquid chromatography. The solubilities and chemical properties of the compounds are different; their pKa values [1] are: vitamin C, 4.04; nicotinamide, 3.3; saccharin, 1.6; vitamin B2 phosphate, pKal, 2.5; pKa2, 6.5; pKa3,10.3; vitamin B6, pKal, 5.0; pKa2, 9.0; and vitamin B1, pKal:, 5.5; pKa2:, 9.5. The objective of this work was to seek rugged combinations of optimized mobile phase conditions for ion-pair reversed-phase liquid chromatography that ensured 634

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complete separation of the water-soluble vitamins in a reasonable analysis time by systematic study of the factors influencing the chromatographic behavior of the WSVs. We proceeded by a sequential approach using experimental design methodology, which enabled modeling and optimization of the chromatographic separation of WSVs. The factors evaluated were the concentration of methanol as organic modifier, the concentration of hexanesulfonate as ion-pair reagent, mobile phase pH and the concentration of triethanolamine (TEtOHA) as competitive base. The relative importance of the four mobile phase parameters and their interactions to the chromatographic behavior of each compound were examined by application of a Draper-Lin small composite design [2, 3]. Retention times and peak-widths at half-height were measured and quadratic polynomial models enabled fitting of the data. A full second-order polynomial model is one of the most useful models for approximating a region of a multifactor response surface. A rationalization of the widespread use of full second-order polynomial models is that they represent a truncated Taylor series expansion of any continuous function, and such models would therefore be expected to provide a reasonably good approximation of the true response surface over a local region of experiment space [4]. For four factors, the quadratic model is of the form:
Y = B 0 + B1X1 + B2X2 + B3X3 + B4X4 + B12X1X 2 + BI3X1X3 + B14X1X4 + B23X2X 3 +

In general the composite design consists of a 2k factorial or a 2k-q fractional factorial portion (called a cube), with runs selected from the 2 k runs (Xl, x2 ...... Xk) = (1, 1,..., 1) usually of resolution V or higher, plus 2k axial points at a distance (x from the origin, plus no center points (0, 0 ..... 0). Composite design are extremely useful for sequential experimentation in which the cube portion enables estimation of the first-order effects and interaction effects, and the addition of star points enables second-order terms to be added to the model and to be estimated. A geometric representation of a three-factor Draper-Lin small composite design is depicted in Figure 1.

Experimental
Samples, Chemicals and Solvents
The reference substances, ascorbic acid (vitamin C), thiamine mononitrate (vitamin B1), pyridoxine hydrochloride (vitamin B6), nicotinamide (vitamin B3) and riboflavin phosphate (vitamin B2), were supplied by Federa (Brussels). Chemicals and solvents were of pro analysis quality: hexanesulfonic acid Na salt.H20, 98 %, was from Janssen Chimica (Beerse, Belgium). Triethanolamine, phosphoric acid, acetic acid, dimethylsulfoxide, potassium dihydrogen phosphate and sodium hydroxide solution were supplied by Merck (Darmstadt, Germany) and methanol was from Lab-Scan (Dublin, Ireland). The water used for HPLC was prepared with a Milli-Q system (Millipore, Milford, MA, USA).

B24X2X~ + B34X3X4 + BllX12 + B22X22 + B33X32 + B44X4 + E R R O R where Y is the measured response variable, X 1 - X 4 are the independent chromatographic variables, BO is the intercept.(or constant term), B1-B4 are the first order coefficients, B12-B34 are the interaction coefficients and Bll-B44 are the (pure) second-order coefficients. In the general case, consider the situation in which a response y depends on k factors, coded as xl, x2 ...... Xk. We can approximate the response function over a limited experimental region by a quadratic response-surface model of the form: k k k Y=13o + ZI3~x~ + E [~iix~ "1- E E ~ i j x i x j "1-~
i=l i=l i>j=l

Apparatus and Column

Liquid chromatography was performed with a Waters model 600 multi-solvent delivery system (MilliporeWaters, Milford, MA, USA) enabling use of a mixed mobile phase from four reservoirs continuously degassed with helium at a flow rate of 25 mL min -1. The pump had a built-in 'silk' feature, a pump transducer feed-back loop enabling the flow to be constantly monitored and adjusted for pulse-free flow. A Waters 996 photo-diodearray detector was linked to an NEC Powermate 386/33i data station. A Marathon autosampler (Spark Holland, Emmen The Netherlands) was provided with a 20 ~tL loop. The 250 x 4.6 mm i.d. Alltima column (Alltech) with packed with C8-bonded 5-~m spherical silica. The flow rate was kept constant at 1 mL min -1. Twodimensional chromatograms were recorded at a wavelength of 270 nm.

The quadratic model has a constant term, k first-order terms, k quadratic terms, k ( k - 1)/2 interaction terms and thus a total o f p = 8 9 + 1)(k + 2) parameters. The specific choice of design to obtain the data for such a model fitting would depend on the relative importance to the experimenter of various design features [5]. In this work we used an appropriate small experimental design of composite form for estimating the 89 parameters, such that the number of runs is as little in excess ofp as possible. Composite design for fitting second-order surfaces were initially suggested by Box and Wilson [6] (who added star designs to 2 k factorials) and followed up by Box and Hunter [7]. 635

Mobile Phase Composition

Four separate aqueous solutions, each containing a component of the mobile phase, were stored in four solvent reservoirs A, B, C and D. Reservoir A contained 100 mM triethanolamine; reservoir B 50 mM hexanesulfonate; reservoir C 50 % (v/v) methanol-water; and reservoir D 50 mM potassium dihydrogen phosphate (KH2PO4). The pH of each solution was previously adjusted with Original

Chromatographia Vot. 44, No. 11/12, June 1997

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Plot of the Draper-Lin small composite design in three dimensions.

Table I. Determination of the parameter space. Chromatographic factor Coded values Triethanolamine (mM) Hexanesulfonate (mM) Methanol (% v/v) pH Low level -1 15 2.5 15 3 Central level 0 20 5 20 4 High level +1 25 7.5 25 5 Low axial level - ct -1.41 12.93 1.46 12.93 2.59 High axial level + cx +1.41 27.07 8.54 27.07 5.41

phosphoric acid or sodium hydroxide solution (1 M) according to the prescribed p H value for the different runs of the design applied. The volumes taken from reservoirs A, B and C by the multi-solvent delivery system were calculated to fulfil the different mobile phase combinations in the design per p H level. Reservoir D was used to top up the total volume to 100 % (v/v). A Draper-Lin small composite design was performed in 18 experiments within the boundaries defined in the par a m e t e r space shown in the Table I. The worksheet of the design in coded values is reproduced in Table II. The retention times and the peak-widths at half-height were measured and recorded as response variables; their values are shown in Tables III and IV. The 18 runs were completed in approximately five days.

water and diluted to volume. This solution (1.0 m L ) was introduced to a 10-mL volumetric flask, D M S O (0.8 mL) was added, the mixture was diluted to volume with the mobile phase prepared according to the run to be performed, and 20 gL of the solution was injected.

Sample solutions
Twenty effervescent tablets were weighted individually to determine the average weight and a portion of powder equivalent to twice the average weight of the tablets was accurately weighed into a 50.0-mL volumetric flask. D M S O (10 mL) was added and the flask put in an ultrasonic bath for approximately 10 rain. Small volumes of 0.0125 M Na-hexanesulfonic acid were added until all the CO2 had completely evolved and the solution was then diluted to volume with 0.0125 M hexanesulfonate solution and centrifuged for 10 min at 2000 rev rain -1. The supernatant (4.0 mL) was transferred into a 10.0 m L volumetric flask, adjusted to volume with mobile phase, and 20 laL of the solution was injected. All the solutions were protected from light.

Sample Preparation

Reference Solutions
Each reference standard (50.0 mg) was accurately weighed into a 50.0-mL volumetric flask, dissolved in Original

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Table II. Applied Draper-Lin small composite design (in coded units). Run Triethanolamine (mM) 0 1 1 1 -1 1 -1 -1 -1 -1.41 1.41 0 0 0 0 0 0
0

Hexanesulfonate (mM) 0 1 1 -1 1 -1 -1 1 -1 0 0 -1.41 1.41 0 0 0 0

0

Methanol (% v/v) 0 1 -1 1 -1 -1 1 1 -1 0 0 0 0 -1.41 1.41 0 0

0

PH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Low level (-1) Central level (0) High level (+1) Level (-c0 Level (+ct)

0 -I -1 1 1 1 -1 1 -1 0 0 0 0 0 0 -1.41 1.41
0

15 20 25 12.93 27.07

2.5 5 7.5 1.46 8.54

15 20 25 12.93 27.07

3 4 5 2.54 5.41

Table HI. Measured values for the response variable retention time (min).

Run 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

B1 9.222 9.910 19.713 4.930 17.463 8.913 4.813 7.628 7.568 9.690 9.120 4.808 15.233 17.652 6.542 8.937 8.475 9.313

B2 7.348 5.130 11.432 5.997 10.045 14.812 6.208 4.582 15.433 7.037 7.987 10.332 6.463 15.562 4.654 8.410 6.832 7.438

B3 5.960 4.813 6.232 5.097 7.478 7.562 4.525 5.065 5.700 5.928 6.170 6.065 5.997 8.112 4.728 4.727 6.015 5.982

B6 6.817 6.460 10.997 4.297 7.447 5.947 4.813 4.762 7.568 7.013 6.853 4.957 8.333 10.868 5.175 7.020 4.925 6.863

C 2.818 3.013 3.165 2.513 2.378 2.528 3.008 2.365 3.183 2.797 2.820 2.882 2.780 2.912 2.745 3.110 2.382 2.827

Saccharin 4.407 3.915 5.528 4.287 4.550 6.163 4.543 3.442 6.798 4.205 4.567 5.650 3.930 5.710 3.657 5.730 4.298 4.397

Ell0 9.897 5.732 14.412 8.620 12.167 26.913 8.910 4.792 27.315 8.927 11.217 19.683 7.347 22.293 5.490 11.147 9.865 10.218

Data Analysis
The statistical analysis of data and multiple regression were performed by the software Statgraphics version 7.0. ( M a n u g i s t i c s , Inc., 2115 E a s t J e f f e r s o n S t r e e t , R o c k v i l l e , M a r y l a n d , U S A , 1993). T h e p r e d i c t i o n s of r e t e n t i o n t i m e s a n d p e a k - w i d t h at h a l f - h e i g h t for v a r i o u s mobile phase conditions were calculated by means of a s p r e a d s h e e t p r o g r a m ( E x c e l v e r s i o n 5.0; M i c r o s o f t ) . This s o f t w a r e was also u s e d to d r a w c o n t o u r plots o f minimum effective resolution and maximum retention t i m e s as f u n c t i o n s o f m o b i l e p h a s e p a r a m e t e r s .

Results and Discussion Estimation of the Main Effects and Interaction Effects
T h e classical d e f i n i t i o n o f t h e effect o f a n i n d i v i d u a l m o bile p h a s e p a r a m e t e r o n t h e r e s p o n s e v a r i a b l e is t h e m e a n ( a v e r a g e ) r e s p o n s e v a l u e at its h i g h levels (+) minus t h e m e a n r e s p o n s e v a l u e at its l o w levels ( - ) . T h e effects o f p a r a m e t e r i n t e r a c t i o n s a r e o b t a i n e d b y s u b t r a c t ing the m e a n v a l u e o f t h e i r p o s i t i v e p r o d u c t s a n d t h e i r Original

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TableIV.
Run 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Measured values for the response variable peak-width at half-height (min). B1 0.255 0.267 0.366 0.138 0.365 0.219 0.166 0.205 0.284 0.312 0.251 0.135 0.323 * 0.182 0.248 0.214 0.268 B2 0.222 0.169 0.265 0.178 0.339 0.390 0.188 0.148 0.449 0.211 0.224 0.283 0.202 0.385 * 0.252 0.207 0.224 B3 0.149 0.144 0.147 0.131 0.180 0.180 0.118 0.130 0.141 0.148 0.161 0.151 0.181 0.198 * 0.118 0.151 0.150 B6 0.158 0.151 0.269 0.126 0.200 0.166 0.166 0.140 0.284 0.161 0.162 0.130 0.191 0.257 0.136 0.158 0.154 0.160 C 0.095 0.085 0.129 0.083 0.086 0.083 0.078 0.083 * 0.099 0.113 0.132 0.107 0.125 0.100 0.134 0.082 0.097 Saccharin 0.116 0.109 0.136 0.112 0.120 0.144 0.119 0.094 0.155 0.112 0.120 0.137 0.107 0.137 0.103 0.138 0.115 0.115 Ell0 0.447 0.215 0.433 0.273 0.358 0.663 0.315 0.168 0.752 0.341 0.460 0.477 0.265 0.597 0.189 0.486 0.459 0.459

*Not measured.

negative products. An estimated effect (h) can be considered as significant if the value zero is not included in the 95 % confidence interval, h + 2o(h) (i.e. the estimated effect + twice its standard error), of the estimated effect calculated by a Student t-test. [8, 9]. If the confidence interval contains zero, the estimated effect is not significant. Calculation of standard error for effects or interactions using replicated runs and for higher order interactions (in the case of no replicates) is discussed by Box et al. [10]. In regression analysis factor and interaction effects are just half the values of classical factor and interaction effects. This can be explained by the classical definition of factor effect which is simply the difference in average response. This definition involves a change in response only, a bare By. The change in factor from low to high level, 5x is ignored. This was the case in early types of designed experiments where researchers (for example in agriculture) usually used nominal or ordinal variables, not variables expressed on interval or ratio scales. In m o d e r n research using interval and ratio scales the 8x should not usually be ignored. Because the variable Xl changed from a coded level of -1 to a coded level of +1, 8xl = 2. Thus the factor effect -- 5y1/Sxl per two coded units and the factor effect per one coded unit from Xl = 0 to Xl =1 (as measured by regression analysis) is smaller by 89 than the result obtained from the classical approach. The m e t h o d of calculation of the main effect and interaction effects, with their standard error, is given by Box et al. [11].

cant or not. Significant parameters are those for which the Fisher variance ratio (F-ratio) is large and expressed by a P-value (significance level) less than 0.05.

Regression Modeling
The retention time and the peak-width at half-height are modeled. Multiple regression models express the mathematical relationship between the measured response variables and the independent variables. If a full factorial design is used to fit a linear quadratic model, three levels for each factor will be necessary. For four factors, the number of experiments is too great (81, ; 34). In this work, however, we used a small composite design with 18 runs only to fit second-order regression equations. We combined a two-level fractional factorial design (or cube portion) 24.1 (eight runs), with a star design - the axial distance ct = 1.414 (eight axial points) plus two center points. Small designs might be appropriate when experimentation is expensive, difficult, or time-consuming. Tables V and VI list the regression equation characteristics calculated for retention time and peak-width at half-height, respectively, of the different compounds; the intercepts and regression coefficients are also included in the tables.

Model Adequacy Checking

The primary diagnostic tool for checking the adequacy of the models calculated is residual analysis. The results revealed a normal distribution of residuals. The sum of squares (SSQ) of absolute deviations between predicted and experimental data, and the average relative deviation between calculated and experimental data (ARD), as illustrated in Table VII, provide some idea of the accuracy of the models. The large SSQ values for retention times are largely because of the magnitude of the parameter.

Analysis of Variance
The theory of analysis of variance is well discussed by Allus et al. [12, 13] Analysis of variance can help determination and confirmation of which factors are signifiOriginal

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Table V. Regression equations obtained for retention time, tR (the estimated coefficients are shown and their standard errors). Compound Vitamin B1 Model equation for retention time (tR) tR = (17.1 + 4.61) + (5.22 + 0.536) B + (-1.93 + 0.388) C + (-0.129 + 0.019) B C + (0.047 + 0.009) C 2 + (-0.299 0.094) B D + (1.188 + 0.506) D tR = (54.75 _+1.55) + (0.041 + 0.014) A +' (-2.498 0.190) B + (-3.12 + 0.138) C + (-0.42 5: 0.068) D + (0.063 + 0.007) B C + (0.068 5: 0.013) B ~+ (0.051 + 0.003) C2 tR = (19.99 + 1.929) + (1.915 0.415) B + (-1.405 + 0.183) C + (0.063 -+ 0.014) A B + (-0.016 -+ 0.004) A C + (-0.064 0.010) B C +(-0.348 0.077) B D + (0.176 0.023) C D + (0.024 5: 0.004) Cz + (-0.302 + 0.053) D 2 tR = (-1.897 + 1.819) + (0.024 + 0.065) C + (4.44 + 0.721) D + (-0.057-+0.016) C D +(-0.352 -+ 0.080) D 2 tR = (4.16 5: 0.256) + (-0.041 0.008) B + (--0.039 _+0.009) C + (-0.093 -+ 0.101) D + (0.001 _+0.000) A B + (0.007 5: 0.002) C D + (-0.043 5:0.011) D 2 tR = (16.53 5: 1.371) + (-0.748 + 0.171) B + (-0.233 + 0.032) C + (-2.461 + 0.603) D + (0.014 + 0.006) B C + (0.024 + 0.012) B2+ (0.262 + 0.075) D 2 tR = (100.45 + 5.2)+ (-8.457 5: 0.660) B + (-5.20 0.479) C +(0.206 + 0.023) B C + (0.259 _+0.046) B z + (0.072 + 0.012) C~

Vitamin B2

Vitamin B6

Vitamin B3 Vitamin C

Saccharin

El10

Table VI. Regression equations obtained for peak-width at half-height, W~ (the estimated coefficients are shown and their standard errors). Compound Vitamin B1 Vitamin B2 Model equation for peak-width at half-height (W,A) W,A = (0.403 + 0.054) + (-0.002 + 0.002) A + (0.022 5: 0.003) B + (-0.011 + 0.002) C W,A = (1.278 5: 0.179) + (-0.051 + 0.015) B + (-0.071 + 0.017) C + 0.002+0.001) B C + (0.001 + 0.000) C 2 W,,~ = (0.885 5: 0.122) + (-0.036 _+0.013) B + (-0.053 + 0.011) C + 0.002 + 0.001) A B + (-0.003 _+0.001) A D + (0.002 + 0.001) C D + (0.001 + 0.000) C 2 W~ = (0.227 + 0.022) + (-0.004 + 0.001) C W~ = (0.292 5: 0.022) + (-0.004 +_0.000) B + (-0.003 + 0.000) C + -0.042 + 0.011) D + (0.004 0.001) D 2 W~ = (1.62 + 0.157) + (-0.121 + 0.029) B + (-0.051 + 0.004) C + (0.004 + 0.001) B C

Vitamin B6

Vitamin B3 Saccharin

El10

Table VII. Accuracy of model descriptions. Parameter tR (min) WI/2 A R D (%) SSQ A R D (%) SSQ B1 4.10 4.831 8.22 0.011 Bz 1.65 0.554 7.02 0.008 B3 2.50 0.671 7.71 0.005 B6 2.36 0.524 6.34 0.004 C 0.71 0.011 Saccharin 2.93 0.494 2.25 0.0002 El10 4.I6 8.005 9.47 0.035

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No value of A R D was recorded for vitamin C because no parameters seem to influence its peak-width in the parameter space examined. The average peak-width at half-height was considered in predictions for vitamin C. The ARD values fall within the interval 0.71--4.16 % for retention times and 2.25-9.47 % for the peak-width at half-height. These intervals demonstrated the adequacy of the models for fitting the data. Diagnostic plots of observed against predicted retention times for vitamins B t, B2, B3, B6 and C, and for saccharin and El10 are reproduced in Figure 2, which demonstrates the powerful ability of the quadratic models to fit the data. Original

Optimization and R u g g e d n e s s o f the M e t h o d s Two response criteria were chosen for optimization of the mobile phase parameters for the separation of compounds: the minimum effective resolution (Rs min) and the maximum retention time. (tR max). The quadratic models obtained enabled the prediction of retention times and the peak-width at half-height for all the compounds under study. The spreadsheet program Excel 5.0 was used for calculation with various sets of conditions imposed on the parameters, at fixed mobile phase pH. The three mobile phase parameters were combined with increments of two units within their examined boundaries, resulting in 225 combinations. The retention times

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tained using the mobile phase combination giving optiTable VIIl. Examples of the values obtained for minimum effective resolution (hexanesulfonate 7.5 mM, pH 3.0). TEtOHA (mM) 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 14 1.33 1.76 2.18 2.11 1.66 1.22 0.79 0.37 0.05 0.46 0.86 1.26 1.65 2.03 2.41 15 2.15 1.96 1.51 1.06 0.63 0.20 0.22 0.63 1.03 1.43 1.82 2.20 2.58 2.71 2.64 Minimum effective resolution, Rs min, for the methanol concentrations (% v/v): 16 1.38 0.93 0.50 0.07 0.35 0.77 1.17 1.57 1.95 2.33 2.48 2.41 2.33 2.26 2.19 17 0.40 0.03 0.45 0.86 1.26 1.66 2.04 2.29 2.21 2.13 2.06 1.98 1.90 1.82 1.75 18 0.49 0.90 1.30 1.69 2.08 2.06 1.98 1.90 1.82 1.73 1.65 1.57 1.49 1.40 1.32 19 1.26 1.65 1.98 1.89 1.80 1.72 1.63 1.54 1.45 1.36 1.28 1.19 1.10 1.01 0.92 20 1.88 1.78 1.69 1.60 1.50 1.41 1.31 1.22 1.13 1.03 0.94 0.85 0.75 0.66 0.56 21 1.66 1.56 1.46 1.36 1.26 1.16 1.06 0.96 0.86 0.76 0.66 0.56 0.46 0.36 0.26 22 1.51 1.40 1.29 1.19 1.08 0.97 0.87 0.76 0.65 0.55 0.44 0.33 0.23 0.12 0.01 23 1.44 1.33 1.22 1.10 0.99 0.88 0.76 0.65 0.53 0.42 0.31 0.19 0.08 0.03 0.15 24 1.49 1.37 1.25 1.13 1.01 0.89 0.77 0.64 0.52 0.40 0.28 0.16 0.04 0.08 0.20 25 0.97 1.20 1.41 1.28 1.15 1.02 0.90 0.77 0.64 0.51 0.38 0.25 0.13 0.00 0.13 26 0.51 0.31 0.11 0.07 0.25 0.42 0.59 0.75 0.90 0.77 0.63 0.50 0.36 0.23 0.09 27 0.52 0.46 0.40 0.35 0.30 0.26 0.21 0.17 0.12 0.08 0.05 0.01 0.03 0.06 0.10

and peak-width at half-height were predicted for each c o m p o u n d . The c o m p o u n d s were then sorted in ascending order of elution and the resolution between successive p e a k s was calculated. Subsequently, the minimum effective resolution and m a x i m u m retention times were selected for each p a r a m e t e r combination. In addition predictions at a fixed hexanesulfonate concentration of 7.5 mM and p H = 3.0. were p e r f o r m e d with one unit increment. A n example of the values of RS min obtained are shown in Table V I I I . Contour plots were constructed for the m i n i m u m effective resolution, Rs rain, and the m a x i m u m retention times were predicted. The c o m m o n sections for mobile phase p a r a m e t e r s where m i n i m u m rugged resolution Rs rain > 1.5 (see ref. 14) and tR m a x was m i n i m u m were selected f r o m the contour plots. The c o m m o n sections show o p t i m u m mobile p h a s e combinations for c h r o m a t o g r a p h i c separation of the c o m p o u n d s examined. Resolution was calculated using the formula Rs = [1.18 ( t R 2 - tR1)] / [W,A(2) + W,A(1)]. According to the contour plot (Figure 3a) of minimum effective resolution as a function of methanol content and triethanolamine concentration at a fixed p H of 3.0 when the hexanesulfonate concentration was 1.5 raM, there are three domains that furnish o p t i m u m results. N e a r the right side there is a domain with Rs rain between 2.00 and 2.50 and another with Rs rain between 1.50 and 2.00; the third o p t i m u m domain, in the middle, has a Rs min of 1.50-2.00. The last is preferable because although the separation quality is slightly lower it is m o r e rugged. It should be stressed that here the ruggedness is a m e a s u r e of a system's lack of sensitivity to small changes in operating conditions. The domains on the right are sensitive to small fluctuations in methanol content. The contour plot of d e p e n d e n c e of m i n i m u m effective resolution on m e t h a n o l and triethanolamine concentrations at p H 3.0 w h e n the hexanesulfonate concentration is 3.5 m M is shown in Figure 3b. The domain on 641

the right gives high quality separation with Rs min between 1.5 and 2.00; this domain is, however, highly p r o n e to variation on small changes in m e t h o d conditions and is, therefore, not rugged. Figure 3c shows the contour plot of m i n i m u m effective resolution as a function of the concentrations of methanol and triethanolamine at p H 3.0 when the concentration of hexanesulfonate is 5.5 raM; no o p t i m u m domain with Rs min > 1.5 was detected. A plot of the d e p e n d e n c e of m i n i m u m effective resolution in hexanesulfonate and triethanolamine concentration when the methanol content is 14 % v/v (Figure 3e) revealed two o p t i m u m domains with Rs min of 2-2.5 and 1.5-2, on the right of the plot. These regions are not robust, however. E x a m i n a t i o n of the c o n t o u r plot of minimum effective resolution against hexanesulfonate and triethanolamine concentration for a m e t h a n o l content of 18 % v/v (Figure 3f) shows two o p t i m u m zones one on the right with Rs min = 2.00-2.50 and 1.50-2.00 and another on the left with Rs min = 1.50-2.00; again neither of the two zones is rugged. The most o p t i m u m and rugged conditions were revealed by exploring the response surface of m i n i m u m effective resolution for a hexanesulfonate concentration of 7.5 m M and a fixed p H of 3.0, as r e p r o d u c e d in Figure 3d. The same mobile phase p a r a m e t e r s were explored with one unit increments, as shown in Figure 4. Two domains show regions with Rs min. between 2.50 and 3.00 and between 2.00 and 2.50 (Figure 4, upper plot). These domains provide markedly better ruggedness. Combination of this contour plot with that of maxim u m retention time (Figure 4, lower) for the same eluent composition, enables selection of the best compromise between rugged mobile phase conditions that ensure complete separation of all the c o m p o u n d s under study, in a reasonable analysis time. Figure 5 shows two examples of the c h r o m a t o g r a m s ob-

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Figure 3
Contour plots of minimum effective resolution as a function of mobile phase compositions at pH 3: (a) hexanesulfonate 1.5 mM; (b) hexanesulfonate 3.5 mM; (c) hexanesulfonate 5.5 mM; (d) hexanesulfonate 7.5 mM; (e) methanol 14 % (v/v); (f) methanol 18 % (v/v).

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Figure 4
Contour plots of minimum effective resolution (left) and maximum retention time (right) as functions of the concentrations of methanol and triethanolamine in the mobile phase (pH 3; hexanesulfonate 7.5 mM).

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Figure 5
Chromatograms showing separation of the water-soluble vitamins in (a) standard solution and (b) effervescent tablet. Column: Alltima 250 x 4.6 mm i.d. 5 pm Cs-bonded spherical silica. Mobile phase: isocratic; methanol, 20 % v/v; hexanesulfonate, 7.5 mM; triethanolamine 15 mM; pH 3 (KH2PO4 buffer); flow 1 mL min -1. PDA detection at 270 nm. Order of elution: ascorbic acid, saccharin, nicotinamide, riboflavin phosphate, sunset yellow FCF, pyridoxine hydrochloride and thiamine nitrate.

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mum separation of the compounds examined. When the chromatograms were recorded 2 months later under the same conditions, i.e. mobile phase methanol 20 %, hexanesulfonic acid 7.5 mM, triethanolamine 15 mM at pH 3.00 (buffer KH2PO4 50 mM), the retention times were a good match with the previous results. This shows that the stability of the chromatographic system is good.

References
[1] A. Albert, E. P. Serjeant, "The Determination of Ionization Constants, A Laboratory Manual", 3rd edn, Chapman and Hall, USA, 1984, p. 167. [2] N. R. Draper, D. J. K. Lin, Technometrics 27, 173 (1985). [3] N. R. Draper, D. J. K. Lin, Technometrics 32, 187 (1990). [4] S. N. Derning, S. L. Morgan, "Experimental Design: A Chemometrics Approach", Elsevier, Amsterdam, 1993,p. 246. [5] G. E. P. Box, N R. Draper, "Empirical Model Building and Response Surfaces", John Wiley and Sons, Inc., New York, 1987, pp. 502-525. [6] G.E.P. Box, K. B. Wilson, J. Roy. Statist. Soc. Ser. B 13, 1 (1951). [7] G. E. P. Box, W. G. Hunter, J. S. Hunter, Ann. Math. Stat. 28, 195 (1957). [8] D. L. M assart, B. G. M. Vandeginste, S. N. Deming, Y. M ichotte, L. Kaufrnan, in "Chemometrics: A Text Book", Elsevier, Amsterdam, 1988, p. 101. [9] G Sado, M. C. Sado, "Les Plans d'Exp6rience; de l'Experimentation ~ l'Assurance de Qualit6", Afnor Technique, Paris, 1991, p. 70. [10] G.E.P. Box, W. G. Hunter, Z S. Hunter, in "Statistics for Experimenters, An Introduction to Design, Data Analysis and Model Building",John Wiley and Sons, Inc., New York, 1978, p. 327. [11] G. E. P. Box, W. G. Hunter, J. S. Hunter in "Statistics for Experimenters, An Introduction to Design, Data Analysis and Model Building,John Wiley and Sons, Inc. New York, 1978, p. 309. [12] M. A. Allus, R. G. Brereton, G. Nickless, Chemometr. Intell. Lab. Syst. 3, 215 (1988). [13] M. A. Allus, R. G. Brereton, G. Nickless, Chemometr. Intell. Lab. Syst. 6, 65 (1989). [14] L. R. Snyder, J. Glajch, J. Kirkland, Practical HPLC Method Development, John Wiley and Sons, Inc., New York, 1988,p. 17. [15] E. Morgan, K. W Burton, P. A. Church, Chemometr. Intell. Lab. Syst. 5, 283 (1989). [16] J.O. De Beer, C. V. Vandenbroucke, D. L. Massart, J. Pharm. Biomed. Anal. 12, 1379 (1994). [17] J. (9. De Beer, C. V. Vandenbroucke, D. L. Massart, B. M. De Spiegeleer, J. Pharm. Biomed. Anal. 14, 525 (1996). [18] E Rouberty, J.Fournier, Chromatographia. 41,553 (1995).

Conclusion
The experimental design methodology [15-18] followed in this work enabled modeling and optimization of the chromatographic separation of the WSVs and the excipient materials saccharin and the dye Sunset yellow ( E l l 0 ) . The Draper-Lin small composite design enabled considerable reduction of the number of experiments without loss of information and accuracy. The dominant parameters affecting retention time and peak-width at half-height were first identified and subsequently fitted to second-order polynomial models (calculated by multiple regression), with good statistical reliability. The accuracy of the models was assessed by evaluation of two criteria: the sum of squares (SSQ) of the absolute deviations between predicted and experimental data, and the average relative deviation between calculated and experimental data (ARD). The A R D (%) between measured and predicted values were 0.71-4.16 % for retention times and 2.25-9.47 % for peak-width at halfheight. Prediction of retention times and peak-widths at half-height were calculated by use of a spreadsheet for 225 different mobile phase combinations within the parameter boundaries examined. The minimum effective resolution (Rs min) and the maximum retention time (tR max) were monitored as objective criteria. The resulting contour plots furnished optimum and rugged combinations of mobile phase parameters that ensured baseline-resolved peaks within a reasonable analysis time. The best conditions can be selected at a glance by considering the threshold minimum effective resolution Rs rain > 1.5 and the minimum analysis time (i.e. tR max has to be minimized).

Received: Nov 21,1996 Revised manuscripts received: Feb 11 and Apr 8,1997 Accepted: Apr 25, 1997

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