Derivative spectrophotometric methods for the determination of clidinium bromide in binary and ternary mixtures.Introduction
Clidinium (CL) is formulated together with either chlordiazepoxide (CH) or Trifluperazine (TR) as binary mixtures in dosage forms which are widely used for the treatment of gastrointestinal and genitourinary tract disorders.
The problem encountered when analyzing these mixtures was the determination of the weakly absorbing, minor component (CL) in the presence of a strongly absorbing major component (CH or TR).
The analysis of multicomponent mixtures without separation of the constituents is rather a difficult task. Derivative spectrophotometry offers enhancement of the qualitative features, thus increasing the UV finger print for the identification of compounds . Furthermore, derivative spectrophotometry is a useful mean of resolving two overlapping spectra and eliminating matrix interference in the assay of two-component mixtures using the zero-crossing technique [2-4].
The derivative-difference spectrophotometry will offer further advantages in canceling heavy spectral interference [5,6]. However, in certain circumstances, the derivative methods cannot cope with the level of interference, a case where the derivative compensation technique is useful [7,8]. This method is used to determine a weakly absorbing minor component (CL) in the presence of a strongly absorbing major component (CH or TR) in their binary mixtures. In two-component analysis, this method is of a value in comparing several difference spectra [mixture (m) reference (r)] using different concentration of a reference solution [Cr= Cx or Cy] in the reference cell. Hence, if Ami and Ari refer to the absorbance of the relevant cells against air at a wavelength i, then [DELTA]Ai =Ami-Ari where Am = Ax + Ay at a given wavelength i, x and y refer to components X and Y, respectively, and Ar refers to Ax or Ay. If Cr for compound X is introduced into the reference cell, the absorption characteristics of the mixture gradually approaches that of compound Y as Cx increases and finally coincides with the absorption curve of compound Y at the balance point, for which Cr =Cx. The accuracy of the method depends on the evaluation of the balance point . therefore, in order to eliminate the personal bias in the detection of the balance point during two-component compensation spectrophotometry, the ratios of derivative measurements as purity indices were adopted [10,11]. The reported methods for the analysis of CL-CH binary mixture were derivative ratio spectrophotometry  and HPLC [13,14]. On the other hand, for CL-TR binary mixture there is no procedure for its analysis has been yet reported.
A derivative spectrophotometric method is developed for the assay of ternary mixture of CL, CH and the degradation product of the latter, 2-amino-5-chlorobenzophenone (ACB).
Salinas et al  introduced a method for resolving binary mixtures which is based on the use of the first derivative of the ratio spectrum. This method and zero-crossing technique have been used to determine ternary mixtures [16-18].
Theoretically, consider a mixture of three compounds M,N and P. If Beer's law is obeyed for all the compounds over the whole wavelength range used and the path-length is 1cm, the absorption spectrum of the mixture is defined by the equation:
[A.sub.m,[lambda]i] = [[epsilon].sub.M, [lambda]i], [C.sub.m] + [[epsilon].sub.N,,[lambda]i] [C.sub.N] + [[epsilon].sub.P,,[lambda]i] [C.sub.P] (1)
Where [A.sub.m, [lambda]i] is the absorbance of the mixture at wavelength [lambda]i, [[epsilon].sub.M,[lambda]i], [[epsilon].sub.N,[lambda]i], and [[epsilon].sub.P,[lambda]i] are the molar absorptivities of M,N, and P at wavelength [lambda]i, and [C.sub.M], [C.sub.N] and [C.sub.P] are the molar concentrations of M,N and P, respectively.
If Eq. (1) divided by the corresponding equation for the spectrum of a standard solution of one of the components (e.g., M of concentration [C.sup.o].sub.M]) and the first derivative of the result is obtained, the following equation can be written:
[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]
This equation indicates that the derivative ratio spectrum of the ternary mixture is independent on the value of [C.sub.M] and dependent of the value of [C.sub.N], [C.sub.P], and [C.sup.o.sub.M] in the ternary mixtures. The content of N and P can be resolved by the zero-crossing method by measuring at adequate wavelengths and by use of calibration graphs.
A calibration graph (e.g. for N) is obtained by recording and storing the spectra of solution of pure N at different concentrations and the spectrum of pure P and the spectrum of a solution of pure M of concentration [C.sup.o.sub.M]. The amplitudes of the spectra of N and P are then divided, wavelength by wavelength, by the corresponding amplitudes for [M.sup.o]. The "ratio spectra" thus obtained are then differentiated with respect to wavelength. The derivative values:
[C.sub.N] d [[[epsilon].sup.N], ,[lambda]i] / d[lambda] [[epsilon].sub.M, [lambda]i] [C.sup.o.sub.M]
are plotted against [C.sub.N] for a given wavelength corresponding to zero-crossing of the ratio of spectrum of P (in this wavelength = d/d[lambda] [[epsilon] p, [lambda] iCp] / [epsilon][lambda] [epsilon] M, [lambda] iC [omicron] M = 0) to give a calibration graph. Application of the method to the sample containing M, N, and P and use of the calibration graph, will then give the value of [C.sub.N] in the ternary mixture.
The spectrophotometric measurements were carried out on a Jasco V-530 double beam UV-VIS spectrophotometer and an Epson LQ-300 printer. The absorption spectra were recorded on the same spectrophotometer, with 1cm quartz cells and supported with Jasco Spectra Manager software for GUL-LIVER Ver.1.53, and a hp Laser Jet 1015 printer.
Materials and reagents
All materials and reagents were of analytical reagent grade. CL was kindly supplied by E.I.P.I. Co., Egypt. CH and TR were kindly supplied by SEDICO pharmaceutical Co., Egypt. ACB was kindly supplied by Aldrich Chem. Co., USA.
For binary mixtures
Stock solutions containing 0.1 mg [mL.sub.-1] CL, 0.2 mg [mL.sub.-1] CH, and 0.5 mg [mL.sub.-1] TR were prepared in distilled water. Further dilutions from each solution were made as described under calibration graphs using distilled water.
For ternary mixture
Stock solutions containing 1.0 mg [mL.sub.-1] CL, 1.0 mg [mL.sub.-1] CH, and 0.5 mg [mL.sub.-1] ACB were prepared in ethanol. Further dilutions were done using 0.1M HCl as described under construction of calibration graphs.
Construction of calibration graphs
For binary mixtures
Various aliquots from CH and TR stock solutions, within the concentration range stated in Table 1, were transferred into two separate sets of 100-mL volumetric flasks, and the solutions were completed to volume with water. The [D.sub.1] and/ or [D.sub.2] spectra of CH and TR solutions were recorded within the wavelength range 200-350nm against water.
The [D.sub.1] and [D.sub.2] values for each solution were recorded at the specified wavelengths (Table 1).
(a) Determination of standard ratios
The measured [D.sub.1] and / or [D.sub.2] values of standard CH and TR solutions at the specified wavelength (Table 1) were used for the calculation of the standard derivative ratios for both drugs.
(b) compensation method
The solution of the mixture containing either CH and CL or TR and CL was placed in the sample cell. Solutions of pure CL of concentration range 2-12 [micro]g [mL.sup.-1] that present in the mixture solution were placed in succession in the reference cell. The [D.sub.1] and / or [D.sub.2] spectra were recorded in each case, and the corresponding ratios (Table 1) were calculated in each instance. The calculated ratios for pure CH or TR (Table 1) were followed to determine the exact balance point (where the ratio of the mixture is equal to that of pure CH or TR). At that point, the concentration of CL in the mixture solution is equal to that in the reference solution.
(c) Graphical method
To avoid the excessive preparation of several standard solutions of CL in locating the exact balance point, a graphical method is recommended. The same steps as for the compensation method were followed and at each time the derivative ratio of the mixture (in the sample cell) is calculated and plotted against the concentration of pure CL in the reference cell (Fig.1). The concentration of CL can easily extrapolated from the graph by substituting the ratio of pure CH or TR.
[FIGURE 1 OMITTED]
For ternary mixture
Into two sets of 100-mL volumetric flasks different aliquots of the standard solutions of CH and CL, within the concentration range in Table 2, were transferred. The solutions were then completed to the volume with 0.1M HCl.
The absorption spectrum of each solution was recorded and stored as data base.
For binary mixtures
Ten tablets from each combination were separately accurately weighed and ground into fine powder. A weight of powder equivalent to 20mg of CH or 50mg TR was transferred into 100-mL volumetric flask, dissolved in water then completed to volume with the same solvent. The flask was shaken for 30 minutes then filtered. The procedures were completed as described under the construction of calibration graphs starting from the beginning.
For derivative ratio method
A weight of powdered tablet equivalent to 50mg of CH and 25mg of CL was transferred into 50mL volumetric flask, dissolved in ethanol then completed to volume with the same solvent. The content of the flask was shaken mechanically for 30 minutes, and then filtered. CH content was determined by completing the procedure on the filtrate as described under calibration graphs. For CL content, different portions from filtrate were transferred into separate 100-mL volumetric flask, then definite quantity of standard CL solution was added to each flask, and the volume of each flask was completed by 0.1M HCl. The measurement was done as described under calibration graphs.
Results and discussion
Preliminary attempts to analyze CL in combination with CH or TR using a direct derivative technique failed to determine CL, because of marked interference of CH or TR in its absorption spectrum (Fig.2 and 3). On the other hand, CH or TR can be determined by direct derivative methods.
[D.sub.2] zero-crossing method
As shown in Fig. 2(c), the [D.sub.2] spectrum of CH exhibited a maximum at 285 nm, while that of CL showed a zero-crossing point at this wavelength.
Similarly, TR can be assayed in the presence of CL by measuring its [D.sub.2] amplitude at 255 nm, where CL shows no contribution [Fig. 3(b)].
Under the experimental conditions described for both mixtures, the graphs obtained by plotting the derivative values versus concentrations for each of CH or TR in the range stated in Table 3 exhibited linear relationships.
Derivative compensation method
The validity of the two-component compensation derivative spectrophotometric method was demonstrated through the assay of CL (minor, weakly absorbing component) in binary mixtures containing CH or TR as a major, strongly absorbing component.
Fig.2 and 3 show the zero order, first and second derivative spectra of CH, TR and CL. The spectrum of either of CH or TR overlapped completely the CL spectrum. Accordingly, the compensation method is proposed for correcting such interference. Therefore, the D1 and D2 maxima for CH and TR were measured and the corresponding derivative ratios were calculated. Table1 shows the mean values of the ratios calculated for six different determinations of CH and TR solutions. The relative standard deviation was less than 1%, indicating good reproducibility. The ratios are constant, characteristic to that of the pure substance, independent of its concentration as well as the presence of another absorbing compound.
For the determination of CL concentration in these mixtures, the sample cell was filled with the mixture solution and the reference cell was filled, in succession, with a serial concentration of CL reference solutions. The ratios of the mixture calculated from the recorded D1 and D2 spectra were compared with those of CH or TR (Table1).
At the exact balance point, the ratio of the mixture corresponds to that of CH or TR, where the concentration of CL in the mixture in the sample cell is equal to that of the reference solution (reference cell).
The graphical method has the advantage of avoiding detailed practical compensation steps. It consists of plotting the calculated ratio of the mixture against the concentration of CL in the reference cell where a straight line is obtained (Fig.1). The concentration of the drug is calculated from the graph, as it is corresponding to the ratio of the mixture concentration (equal to the ratio of either CH or TR in the mixture).
In order to prove the validity and applicability of the proposed method, laboratory made mixtures were prepared in concentration ranges similar to their pharmaceutical preparations; 0.1-0.6[micro]g [mL.sup.-1] CL with 1.0 [micro]g [mL.sup.-1] CH for CH-CL mixture and 0.15-0.40 [micro]g [mL.sup.-1] TR for TR-CL mixture; both above and below the normal levels expected in the dosage forms and analyzed using the described procedure. The results obtained in this study (Table 4 and 5) were good indicating high accuracy and good precision of the proposed method.
[FIGURE 2a OMITTED]
[FIGURE 2b OMITTED]
[FIGURE 2c OMITTED]
[FIGURE 3a OMITTED]
[FIGURE 3b OMITTED]
The ternary mixture containing CL, CH and ACB represents a combination of a weakly absorbing compound (CL) and a strongly absorbing compounds (CH, ACB). The absorption spectrum of CL is completely overlapped by those of CH and ACB (Fig.4). Practical trials were made for the use of the direct and differential derivatives as well as derivative compensation techniques but proved their non-accuracy for CL quantitation. Preliminary attempts to analyze this mixture using derivative ratio method failed to determine CL, partially because of the low concentration and partially because of its weak absorptivity. Therefore, it was suggested that the standard addition of pure CL to this mixture and to the tablets will solve this problem. Thus, the derivative ratio method and the zero-crossing technique was then proposed for this case. On the other hand, CH due to its high contribution in this mixture and its strong absorption in UV region as shown in Fig.4, it can be determined by the use of derivative technique (D2) at zero-crossing point.
[FIGURE 4 OMITTED]
As shown in Fig.5, the [D.sub.2] spectrum of CH exhibited these maxima at 247, 279 and 344 nm, while that of CL and ACB showed zero-crossing points at three wavelengths. Thus, CH could be determined in the presence of CL and ACB by measuring its D2 responses at the above mentioned wavelengths.
[FIGURE 5 OMITTED]
For the determination of CL in this mixture, the stored absorption spectra data of standard solution of CL,CH and ACB and a solution of their mixture were divided (amplitude by amplitude at the appropriate wavelengths) by the absorption spectrum of a standard CH solution. Then the first derivative of the obtained ratio spectra ([sup.1]DD) were calculated with [DELTA][lambda] =1 nm (Fig.6). As shown from this figure, CL can be determined in this mixture by measuring the amplitude at 224 nm where there is no contribution from ACB (zero-crossing point of ACB).
A study was carried out to test for the effect of the divisor concentration on the calibration graphs of CL. Thus, the absorption spectra of standard solutions of CL of different concentrations (Table 6) were obtained. The amplitudes of these solutions were divided by the corresponding amplitudes of standard solutions of CH of different concentrations (Table6). The resultant ratio spectra were then differentiated with respect to wavelength using [DELTA][lambda]=1 nm. The derivative values were measured at 224 nm and plotted against its concentration. A straight line was obtained in each case. The statistical analysis of these graphs using least squares method (Table 6) showed values of correlation coefficients approaching 1.0 and small values of intercepts which indicated good linearity. The results obtained from these data (Table 6) indicated that, the divisor concentration had no effect on the assay. If the concentration of the divisor was increased or decreased, the resulting derivative values were proportionately decreased or increased, although the maxima or minima remain at the same wavelength.
[FIGURE 6 OMITTED]
Validation of the methods
The linearity of the response (D2 values for CH or TR and [sup.1]DD values for CL) as a function of drug concentration was evaluated for each component (Tables 2 and 3). A straight line was obtained in each case. Statistical analysis of these graphs showed excellent linearity of the calibration graphs and with small intercept values, as shown in Tables 2 and 3. This is confirmed through the calculated t = a/Sa (the values did not exceed the theoretical t values at the 95% criterion).
Method selectivity was detected by preparing different mixtures, within the linearity range, such that the mixture contains variable amounts of one component and constant amounts of the other components. The assays were performed according to the previously stated conditions. The corresponding D2 values for CH and TR and [sup.1]DD values for CL were measured at the specified wavelengths in the absence and presence of the other components of the mixtures (Tables 3 and 7). Statistical analysis of these data showed that, the slope of the calibration graph for each drug is independent on the concentration of the other components (Table 3 and 7). The results obtained showed that the [D.sub.2] and [sup.1]DD amplitudes for CH, TR and CL were proportional only to the assigned drug concentration at the specified wavelength. Consequently, the results obtained verified the high selectivity of the proposed methods and reflected the selectivity for the simultaneous determination of the mixtures.
To test the precision of the proposed methods, separate six determinations for each drug alone and in presence of the other components in the mixtures were carried out. The results obtained showed that the coefficient of variation is less than 2% which indicates high precision of the proposed methods (Tables 8 and 9).
In order to test the accuracy of the proposed method, laboratory made mixtures of each combination were prepared in different proportions. The resulting mixtures were assayed according to the above stated procedures and the results calculated as the percentage of analyte recovered. The good recovery values indicated the high accuracy of the proposed methods (Tables 8 and 9).
Assay of dosage forms
The proposed methods were applied to determine the components of these mixtures in dosage forms. The results obtained (Tables 10 and 11) were precise and accurate. This indicated the high applicability for adopting these methods to the assay of such drug combinations in various matrices.
The proposed methods are well-suited to the reliable analysis of dosage forms containing CH,TR with CL. More important, the last described procedure is considered a stability indicating since the degradation product (ACB) did not interference during the quantitation of the two components CH and CL. The most striking features of the derivative technique are its simplicity, sensitivity and rapidity, which render it suitable for routine analysis in quality control laboratories.
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Amira F. El-Yazbi *, Azza A. Gazy and Mahmoud A. El-Sayed
Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Alexandria University, Egypt.
* Corresponding author: E mail: email@example.com
Address: 307 RH Michener Park, Edmonton, Alberta, Canada. Postal code: T6H 4M5.
Table 1: Ratios of the derivative modes at the specified wavelengths for CH and TR. Drug Parameter Mean (a) RSD% CH D1 (273nm)/D1 (234nm) 1.344 0.022 2-12 [micro]g [mL.sup.-1] D1 (273nm)/D2 (285nm) 2.885 0.017 TR D2 (255nm)/D2 (270nm) 1.699 4.43x[10.sup.-3] 5.0-17.5 [micro]g [mL.sup.-1] (a) Mean of six separate determinations Table 2: Assay parameters for the determination of CH and CL in ternary mixture. Drug Conc. range Method [lambda] (mg 100 (nm) [mL.sup.-1]) CH 0.2-1.0 [D.sub.2] 247 279 344 CL 2.0-12.0 [sup.1]DD 224 Linear regression Drug Intercept (a) Slope (b) Corr. Coeff. (r) CH -3.93x[10.sup.-6] 2.10x[10.sup.-3] 0.9999 2.47x[10.sup.-7] 1.26x[10.sup.-3] 0.9999 -9.93x[10.sup.-7] 3.76x[10.sup.-4] 0.9998 CL 2.35x[10.sup.-4] 0.079 0.9999 Standard deviation of Drug Slope Intercept t = a/ ([S.sub.b]) ([S.sub.a]) [S.sub.a] (a) CH 1.61x[10.sup.-5] 1.03x[10.sup.-5] 0.38 2.84x[10.sup.-6] 1.82x[10.sup.-6] 0.13 3.43x[10.sup.-6] 2.19x[10.sup.-6] 0.45 CL 4.10x[10.sup.-4] 3.16x[10.sup.-3] 0.08 (a) Theoretical t values = 2.45 for 95% confidence levels Table 3: Analytical and statistical parameters for the assay of CH and TR in binary mixtures containing CL. Sample (conc. mg [lambda] [mL.sup.-1]) nm CH CL 0.20-1.20 -- 285 0.20-1.20 0.50 TR CL 0.50-1.75 -- 255 0.50-1.75 0.25 Regression equations Sample (conc. mg Intercept Slope Corr. Coeff. CV% (a) [mL.sup.-1]) (a) (b) (r) CH CL 4.8x[10.sup.-3] 0.9999 1.89 0.20-1.20 -- 0.48386 0.9996 0.96 0.20-1.20 0.50 1.51x[10.sup.-2] 0.47171 TR CL 1.55x[10.sup.-3] 0.9999 0.51 0.50-1.75 -- 0.39966 0.9998 1.49 0.50-1.75 0.25 1.61x[10.sup.-3] 0.39783 (a) Mean of six experiment Table 4: Assay results for laboratory made mixtures containing CH and CL. Component [D.sub.1] [D.sub.1] % Recovery conc. (273nm) (273nm) ([micro]g R R [mL.sup.-1] [D.sub.2] [D.sub.2] [R.sub.1] [R.sub.2] (243nm) (285nm) CH CL ([R.sub.1]) ([R.sub.2]) 1.0 0.1 1.355 2.881 100.81 99.86 1.0 0.2 1.356 2.873 100.94 99.59 1.0 0.3 1.350 2.861 100.44 99.17 1.0 0.4 1.348 2.871 100.36 99.51 1.0 0.5 1.342 2.2.884 99.91 99.97 1.0 0.6 1.341 2.869 99.81 99.43 Mean 100.38 99.59 [+ or -] SD [+ or -] 0.46 [+ or -] 0.29 Table 5: Assay results for laboratory made mixtures containing TR and CL. Component conc. [D.sub.2] (255nm) % Recovery (mg [mL.sup.-1]) R [D.sub.2] (270nm) TR CL 1.5 0.15 1.703 100.20 1.5 0.20 1.699 99.97 1.5 0.25 1.696 99.80 1.5 0.30 1.698 99.92 1.5 0.35 1.695 99.97 1.5 0.40 1.699 99.99 Mean [+ or -] SD 99.94 [+ or -] 0.15 Table 6: Effect of divisor concentration on the determination of CL by derivative ratio method. Divisor CL conc. CH conc. (mg 100 (mg 100 [mL.sup.-1]) [mL.sup.-1]) Linear regression Intercep Slope Corr. t (a)x (b) Coeff. [10.sup.4] (r) 2.0-12.0 0.2 40.70 0.223 0.9999 0.5 2.35 0.079 0.9998 0.6 -8.00 0.071 0.9999 Divisor CL conc. CH conc. (mg 100 (mg 100 [mL.sup.-1]) [mL.sup.-1]) Standard deviation of Slope Intercept t=a/ ([S.sub.b])x ([S.sub.a])x [S.sub.a] [10.sub.4] [10.sub.3] (a) 2.0-12.0 0.2 6.78 5.28 0.77 0.5 4.10 3.16 0.07 0.6 3.44 2.68 0.30 (a) Theoretically t values = 2.45 for 95% confidence levels Table 7: Assay parameters for the analysis of CH and CL in ternary mixtures by the proposed methods. Mixture conc. (mg 100[mL.sup.-1]) [lambda] CH CL ACB Method (nm) 0.2-1.0 -- -- 247 0.2-1.0 0.25 0.2 [D.sub.2] 247 0.2-1.0 -- -- 279 0.2-1.0 0.25 0.2 [D.sub.2] 279 0.2-1.0 -- -- 344 0.2-1.0 0.25 0.2 [D.sub.2] 344 -- 2.0-12.0 -- 224 0.5 2.0-12.0 0.2 [sup.1]DD (a) 224 Linear regression Corr. Intercept Slope coeff. (a) (b) (r) CV% (b) -3.93 x [10.sup.-6] 2.10 x [10.sup.-3] 0.9999 0.78 5.11 x [10.sup.-6] 2.07 x [10.sup.-3] 0.9999 0.82 2.47 x [10.sup.-7] 1.26 x [10.sup.-3] 0.9999 0.33 -1.09 x [10.sup.-7] 1.27 x [10.sup.-3] 0.9999 0.70 -9.93 x [10.sup.-7] 3.76 x [10.sup.-4] 0.9998 0.82 9.61 x [10.sup.-7] 3.70 x [10.sup.-4] 0.9999 0.54 2.35 x [10.sup.-4] 0.079 0.9999 0.77 2.00 x [10.sup.-3] 0.079 0.9998 0.79 (a) Divisor is a solution of CH 0.5 mg% (b) CV% for 6 separate determinations Table 8: Assay results for the determination of CH and TR in laboratory made mixtures with CL Compound Laboratory made mixture Mean recovery determined (mg [mL.sup.-1]) (% [+ or -] SD) (a) CH CH CL 0.20-1.20 0.50 100.64 [+ or -] 0.98 1 0.10-0.60 99.83 [+ or -] 0.42 TR TR CL 0.50-1.75 0.25 99.52 [+ or -] 1.49 1.50 0.15-0.40 99.38 [+ or -] 0.27 (a) Mean of six separate determinations Table 9: Assay results for the determination of CH and Cl in presence of ACB in laboratory made mixtures. Conc. Method [LAMBDA] Mean recovery (mg 100[mL.sup.-1]) (nm) [+ or -] SD (a) CH CL ACB 0.2-1.0 0.25 0.2 [D.sub.2] 247 100.15 [+ or -] 0.82 (b) 0.2-1.0 0.25 0.2 [D.sub.2] 279 99.95 [+ or -] 0.70 (b) 0.2-1.0 0.25 0.2 [D.sub.2] 344 100.02 [+ or -] 0.54 (b) 0.5 2.0- 0.2 [sup.1]DD 224 100.07 [+ or -] 0.79 (c) 12.0 (a) Mean of six separate determinations of CH(b), CL(c). Table 10: Assay results for the determination of CH-CL and TR-CL combinations in dosage forms. Proposed Recovery [+ or -] RSD % (a) Method CH CL [D.sub.2] (285 nm) 99.73 [+ or -] 1.89 -- [D.sub.1] (273nm) [D.sub.1] (234 nm) -- 100.00 [+ or -] 0.46 [D.sub.1] (273 nm) [D.sub.2] (285 nm) 99.99 [+ or -] 0.29 [D.sub.2] (255nm) -- -- [D.sub.2] (255 nm) [D.sub.2] (270nm) -- -- Proposed Recovery [+ or -] RSD % (a) Method TR CL [D.sub.2] (285 nm) -- -- [D.sub.1] (273nm) [D.sub.1] (234 nm) -- -- [D.sub.1] (273 nm) [D.sub.2] (285 nm) -- -- [D.sub.2] (255nm) 99.97 [+ or -] 0.51 -- [D.sub.2] (255 nm) [D.sub.2] (270nm) -- 99.94 [+ or -] 0.15 (a) Mean of six separate determinatins Table 11: Assay results for the determination of CH and CL in dosage forms. Compound Labelled Conc. Method [lambda] determined conc. standard (nm) (mg 100 added [mL.sup.-1]) (mg 100 [mL.sup.-1]) CH 0.20-1.00 -- [D.sub.2] 247 [D.sub.2] 279 [D.sub.2] 344 CL 0.25 4.00 [sup.1]DD 224 0.50 4.00 [sup.1]DD 224 1.00 4.00 [sup.1]DD 224 Compound Labelled Mean determined conc. recovery % (mg 100 [+ or -] SD (a) [mL.sup.-1]) CH 0.20-1.00 99.78 [+ or -] 0.53 100.56 [+ or -] 0.56 100.01 [+ or -] 0.85 CL 0.25 100.15 [+ or -] 0.42 0.50 100.45 [+ or -] 0.42 1.00 99.97 [+ or -] 1.20