H-Point Curve Isolation Method for Coupled Liquid Chromatography

Anal. Chem. 2000, 72, 2559-2565

H-Point Curve Isolation Method for Coupled Liquid Chromatography and UV-Visible Spectrophotometry F. Blasco-Go´mez, F. Bosch-Reig, P. Campı´ns-Falco´,* C. Molins-Legua, and R. Herra´ez-Herna´ndez

Departament de Quı´mica Analı´tica, Facultad de Quı´mica, Universidad de Valencia, C/Doctor Moliner 50, Burjassot, Valencia, E-46100 Spain

The H-point curve isolation method (HPCIM) for the detection of unknown interferences in chromatography is proposed. The method allows one to estimate the UVvis spectra of interfering species in a sample as well as to test the purity of the chromatographic peaks. Besides the detection of the unknown interferences in a sample, this method allows one to calculate the concentration of an analyte in the presence of unknown compounds. To illustrate the reliability of the proposed method, samples of diuretics and amphetamines have been analyzed by normal- and reversed-phase high-performance chromatography. At present, a great number of reports can be found in the literature dealing with the problem of overlapping peaks in chromatography.1-5 The resolution of overlapped peaks depends on the degree of chromatographic overlap and on the spectral features of the coeluting compounds. The greatest challenge is to resolve the total chromatographic overlap of compounds having very similar spectra. Since the presence of a unique peak in a chromatogram does not guarantee the total separation of the compounds in a sample, many researchers have developed methods to check peak purity.6-8 The detection of overlapping peaks is of special importance when the number of peaks in a chromatogram is unknown, as occurs with biological samples or with synthetic products when impurities are present. In research analysis, peak purity information is important. If the peak of one compound is hidden under the peak of another and thus not detected, essential information might be lost. On one hand, impurities hidden under the sample compound falsify the results. On the other hand, the detection of impurities is * Corresponding author. Tel: +34-96-3983002. Fax: 34-96-3864436. E-mail: [email protected]. (1) Kowalski, B. R.; Osten, D. W. Anal. Chem. 1984, 56, 991. (2) Sa´nchez, E.; Kowalski, B. R. Anal. Chem. 1986, 58, 496. (3) Li, J. Anal. Chem. 1997, 69, 4452. (4) Faigle, J. F.; Poppi, R. J.; Scarminio, I. S.; Bruns, E. E. J. Chromatogr. 1991, 539, 123. (5) Bosch-Reig, F.; Campı´ns-Falco´, P.; Verdu´-Andre´s, J. J. Chromatogr., A 1996, 726, 57. (6) Vanslyke, S. J.; Wentzell, P. D. Anal. Chem. 1991, 63, 2512. (7) Fabre, H.; Le Bris, A.; Blanchin, M. D. J. Chromatogr., A 1995, 697, 81. (8) Polster, J.; Sauerwald, N.; Feucht, W.; Treutter, D. J. Chromatogr., A 1998, 800, 121. 10.1021/ac990649q CCC: $19.00 Published on Web 04/21/2000

© 2000 American Chemical Society

important for qualitative studies (i.e., detection of synthetic impurities in a drug substance during the development process,9 studies of metabolites produced by microorganisms or any other kind of cells,10-12 or studies of degradation products during different processes,13 among others). Thus, the detection and identification of all the compounds present in a sample can be as important in biological studies as in industrial processes. The identification of unexpected compounds can help one to understand biological processes and, in industry, to determine the origin of impurities in the final products. There are different procedures that can be used for the identification of unknown substances. The most successful and general approach involves analytical separation followed by a spectroscopic identification. GC/MS and LC/MS14,15 are most frequently employed for this purpose. The use of LC/DAD has also been proposed, where the identification of unknown compounds is made by means of the UV-vis spectra obtained at the top of the chromatographic peaks.16,17 The latter method is useful when the main characteristics of the molecules to be determined are known (e.g., aromatic compounds with different radicals) because the information provided by a UV-vis spectrum is not sufficient to identify a molecule without additional information. The final step for the identification of an unknown compound is a library search for a spectrum that matches the one obtained from the chromatographic analysis.17,18 In a previous paper,19 we proposed a method for the detection of impurities in a sample and for the calculation of their UV-vis spectra based on the measurement of the spectrum of the sample and the spectrum of the analyte. The method cancels the (9) Miller, L.; Bergeron, R. J. Chromatogr., A 1994, 658, 489. (10) Smedsgaard, J. J. Chromatogr., A 1997, 760, 264. (11) Cho, S. C. J. Chromatogr., B 1996, 678, 344. (12) Van Tellingen, O.; Beijnen, J. H.; Van der Woude, H. R.; Bruning, P. F.; Nooyen, W. J. J. Chromatogr., B 1990, 94, 135. (13) Zhang, C. X.; Sun, Z. P.; Ling, D. K.; Zhang, Y. J. J. Chromatogr. 1992, 627, 281. (14) Louter, A. J. H.; Hogenboom, A. C.; Slobodnik, J.; Vreuls, R. J. J.; Brinkman, U. A. Th. Analyst 1997, 122, 1497. (15) Hua, Y.; Lu, W.; Henry, M. S.; Pierce, R. H.; Cole, R. B. J. Chromatogr., A 1996, 750, 115. (16) Bartolome´, B.; Herna´ndez, T.; Bengoechea, M. L.; Quesada, C.; Go´mezCordove´s, C.; Estrella, I. J. Chromatogr., A 1994, 723, 19. (17) Hill, D. W.; Kelley, T. R.; Langner, K. J. Anal. Chem. 1987, 59, 350. (18) Sturaro, A.; Parvoli, G.; Doretti, L. Chromatographia 1994, 38, 239. (19) Blasco-Go´mez, F.; Campı´ns-Falco´, P.; Bosch-Reig, F.; Guomin, L. Analyst 1998, 123, 2857.

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contribution of the analyte signal in the global signal registered, thereby giving a set of possible spectra for the interference. From this set, the real interference spectrum can be calculated by finding pairs of wavelengths according to the calibration model of the H-point standard addition method (HPSAM).20,21 The HPSAM analytical signal (absorbance increment at two wavelengths with the same absorbance value for the interference) is only dependent on one species because at each point the absorbances of the other compounds present in the sample are canceled; if matrix effects are absent, standard additions are not needed and molar absorption coefficients can be used in the HPSAM equation. After the calculation of the interference spectrum, the concentration of the analyte can be calculated by difference. In this work, we propose the application of this method, now named the H-point curve isolation method (HPCIM), to testing the purity of chromatographic peaks. In cases where the peaks obtained are not pure, the method can be also employed to calculate the concentration of the analyte. To asses the features of the method, several samples of amphetamines and diuretics have been analyzed, by normal- and reversed-phase chromatography. THEORETICAL BACKGROUND Consider a mixture of X and Y, X being the analyte and Y being the unknown interfering species. The absorbance of the sample at any wavelength will be the absorbance of the analyte plus the absorbance of the unknown interfering species, so we can write

si ) xi + yi

(1)

where si is the absorbance of the sample at the ith wavelength, the subscript i refers to the absorbance at a certain wavelength in the [1, n] range, and xi and yi are the absorbances of the analyte and the unknown interfering species, respectively, at the ith wavelength. After the selection of a reference wavelength from the X spectrum, we can define Ki,ref as

Ki,ref ) xref/xi

(2)

where xref is the absorbance of the analyte at the reference wavelength. The analyte contribution to the signals can be canceled by the subtraction sref - Ki,refsi to obtain

sref - Ki,refsi ) yref - Ki,refyi

(3)

a result that is related only to the interference. The previous equation can be rewritten in the following way:

yi )

(sref - Ki,refsi) - yref sref - yref ) si -Ki,ref Ki,ref

(4)

This expression allows us to calculate the interference spectrum by entering an approximate value of yref, since everything in the (20) Bosch-Reig, F.; Campı´ns-Falco´, P. Analyst 1988, 113, 1011. (21) Campı´ns-Falco´, P.; Verdu´-Andre´s, J.; Bosch Reig, F.; Molins Legua, C. Anal. Chim. Acta 1995, 302, 323.

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Figure 1. Hypothetical spectra for Y obtained varying the interference absorbance at the reference wavelength between the maximum and minimum predicted values. Wavelengths m and n represent one of several pairs of wavelengths at which ym ) yn.

equation is known except this value. The values of K can be calculated from a spectrum of the analyte and the values of sref Ki,refsi from the spectrum of the sample. Since it is only necessary to work with a pure standard and a sample solution, the selection of the reference wavelength is arbitrary. This selection is based on the standard deviation of yref computed at the reference wavelength as explained below. If the standard additions method is followed, all measured wavelengths can be assayed as possible reference wavelengths. The wavelength that provides the greatest number of sref - Ki,refsi values with a minimum standard deviation (n ) number of processed samples) is selected. It should be noted from eq 2 that Ki,ref is independent of path length and concentration. The next step is to estimate the yref value. The range of absorbances within which yref is included can be easily known. The maximum value of the interference absorbance at the reference wavelength cannot be higher than the absorbance of the sample at this wavelength, and the minimum value is the smallest absorbance value that provides non-negative absorbance values for the interference spectrum at any wavelength calculated from eq 4. We can plot several interference spectra from eq 4 considering the range described by the minimum and maximum absorbance values previously calculated and varying the interference absorbance at the reference wavelength within this range to produce meaningful spectral differences from one spectrum to the next one (see Figure 1). The real unknown interference spectrum will be one of the plotted spectra. To calculate the absorbance of the interference at the reference wavelength yref, it is necessary to find a pair of wavelengths m and n in the [1, j] range with the same absorbance value in every hypothetical interference spectrum (Figure 1) to follow the HPSAM calibration model;20,21 this absorbance value can be different in magnitude from one spectrum to the other (ym ) yn for each calculated spectrum). Different (m, n) pairs of wavelengths can be found as can be derived from Figure 1. A similar figure could be constructed using each wavelength in the range 1 to j as the reference wavelength. To ensure the accuracy of the prediction of the interference absorbance at the reference wavelength, the absorbance of the analyte at the selected wavelengths should be as different as possible.

To facilitate the process of locating the wavelength pairs with the same value of absorbance for all the interference spectra, it is sufficient to check the maximum and minimum spectra since they are the most different (as is shown in Figure 1). If it is possible to find a pair of wavelengths with the same value of absorbance in these spectra, the absorbances at these wavelengths in the remaining spectra will also be equal, as can be derived from Figure 1. Having set ym ) yn in all the interference spectra, we have three equations and three unknowns:

sref - Km,refsm ) yref - Km,refym sref - Kn,refsn ) yref - Kn,refyn

(5)

ym ) yn

So the real value of yref can be calculated from

yref ) sref - Km,refsm + Km,ref[(sref - Km,refsm) - (sref - Kn,refsn)] (6) Kn,ref - Km,ref

Since this equation is only dependent on known quantities, it is then possible to estimate the unknown spectrum by using eq 4. EXPERIMENTAL SECTION Apparatus. A Hewlett-Packard model 1040A liquid chromatograph, equipped with a diode array detector linked to a Chemstation data system (Hewlett-Packard, Palo Alto, CA) was used for data acquisition and storage. The system was coupled to a quaternary pump (Hewlett-Packard, 1050 Series) with a 25 µL sample loop injector for diuretics and for amphetamines in normalphase chromatography and with a 20 µL sample loop injector for amphetamines in reversed-phase chromatography. The detector (Hewlett-Packard, 1100 series) was set to collect a spectrum every 640 ms (every 4 nm over the range 200-600 nm). All the assays were done at ambient temperature. Reagents. All reagents were of analytical grade. Methanol (Scharlau, Barcelona, Spain) and acetonitrile (J. T. Baker, Deventer, The Netherlands) were of HPLC grade. The following reagents were used: amphetamine sulfate (Sigma, St. Louis, MO), methamphetamine hydrochloride (Sigma), β-phenylethylamine hydrochloride (Sigma), 1,2-naphthoquinone4-sulfonic acid sodium salt (Sigma), bumetanide (Boehringer Ingelheim), and ethacrynic acid (Sigma). Sodium hydroxide and sodium chloride (Panreac, Barcelona, Spain), phosphoric acid (Probus, Badalona, Spain), sodium dihydrogen phosphate monohydrate (Merck, Darmstadt, Germany), sodium hydrogen carbonate (Probus), ammonium hydroxide (Probus), hydrochloric acid (Probus), acetate trihydrate (Probus), acetic acid (Probus), and propylamine hydrochloride (Fluka, Buchs, Switzerland) were also used. Water was distilled, deionized, and filtered through 0.45 µm nylon membranes (Teknokroma, Barcelona, Spain). Columns and Mobile Phases. The column for the determination of diuretics was an HP-Hypersyl ODS-C18 column (5 µm, 250 mm × 4 mm i.d.). A 0.05 M phosphate buffer (pH 3)-

acetonitrile gradient with an acetonitrile content increasing from 15% at the start to 80% at 8 min was used as the mobile phase. The flow rate was set at 1 mL/min. A LiChrospher Si-60 (5 µm, 125 mm × 4 mm i.d.; Merck) column was used for the separation of amphetamines in normalphase chromatography. The mobile phase was ethanol-chloroform-ethyl acetate-n-hexane (1:22:32:45 v/v). The flow rate was set at 2 mL/min. For the separation of amphetamines in reversed-phase chromatography, a Hypersil ODS column (5 µm, 250 mm × 4 mm i.d.) was used. The mobile phase was acetonitrile-water with an increasing acetonitrile content: 40% acetonitrile at zero time, 50% at 2.5 min, 70% at 3 min. After 3 min, the percentage of acetonitrile was kept constant. The water solution was prepared by adding 2.5 mL of propylamine in 500 mL of water. This solution was prepared daily. The flow rate was set at 1 mL/min. All solvents were filtered with 0.45 µm nylon membranes (Teknokroma) and degassed with helium before use. Preparation of Solutions. Standard stock solutions of diuretics (1000 µg/mL) were prepared in methanol. Working solutions were prepared by dilution of the stock solutions with distilled and deionized water. Phosphate buffer (0.05 M) for bumetanide-ethacrynic acid mixtures was prepared daily by dissolving sodium dihydrogen phosphate monohydrate in water, the solution being 0.03 M in propylamine hydrochloride. The pH was adjusted to 3 by adding the minimum amount of concentrated phosphoric acid. The standard solutions of amines were prepared by dissolving 100 mg quantities of the pure compounds in 100 mL portions of water. Working solutions were prepared by dilution of the stock solutions with distilled, deionized water. The 1,2-naphthoquinone4-sulfonic acid (NQS) stock solution (0.5% w/v) was prepared freshly for each experiment. The bicarbonate solution was prepared by dissolving 8 g of sodium hydrogen carbonate in 100 mL of distilled water. All solutions were stored in the dark at 2 °C. Derivatization of Amphetamines. (a) Normal-Phase Chromatography. Derivatization with NQS was performed as follows. Different volumes of the stock solutions of the amines were added to 0.5 mL of bicarbonate solution (8% w/v), 0.5 mL of NQS, and distilled water up to 1.5 mL. Each mixture was heated at 70 °C for 20 min. After cooling, the mixture was shaken with the same volume of chloroform for 2 min, followed by centrifuging for 5 min at 1500g. The aqueous phase was discarded, and sulfate anhydrous sodium was added to the organic solution to remove the water. The chloroform layer was filtered through 0.45 µm nylon filters from Teknokroma. (b) Reversed-Phase Chromatography. For the reversedphase analysis, the derivatization procedure was performed in C18 cartridges. The cartridges were previously conditioned by adding 1 mL of methanol, followed by 1 mL of bicarbonate buffer (1%) at pH 10. Next, 2 mL of the samples (water or urine previously spiked with analytes) containing different amine concentrations were transferred to the columns and washed with 3 mL of distilled water. The following derivatization procedure was carried out. NQS reagent (1%), 0.5 mL, was run with 0.5 mL of bicarbonate solution (1%) at pH 10 through the columns that contained the analytes. After 10 min at room temperature, the columns were Analytical Chemistry, Vol. 72, No. 11, June 1, 2000

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Figure 2. Chromatograms obtained at 280 nm, under reversedphase conditions: (A) standard solution of 6 ppm of amphetamine (Amp); (B) urine sample spiked with 6.6 ppm of amphetamine. β-Phenylethylamine (β-Phe) was used as the internal standard.

washed with 3 mL of distilled water. Finally, the reaction products (amine-NQS) were eluted from the columns with 1 mL of acetonitrile-water (1:1). The urine sample was treated as follows. A 0.4 mL aliquot of bicarbonate buffer (8%) at pH 10 was added to 2 mL of the urine sample, spiked or unspiked. The mixture was centrifuged at 1500g for 2 min. Finally, 2 mL of the urine sample was taken from the clear liquid obtained and run through the column. RESULTS AND DISCUSSION Assessing the Purity of the Chromatographic Peaks. In the present work, several determinations were studied. The first one was the determination of amphetamine in urine by reversedphase chromatography. Figure 2 shows the chromatogram of a standard solution containing 6 ppm of amphetamine in water (A) and the chromatogram of a sample of urine spiked with 6.6 ppm of amphetamine (B). As can be seen in chromatogram B, a single peak with a retention time of 3.9 min was obtained. This retention time is consistent with the retention time of the amphetamine in the standard. However, this does not mean that the obtained peak is pure because a urinary endogenous compound could be eluting very close to the amphetamine. In the chromatograms can also be observed a peak at t ) 3.6 min corresponding to β-phenylethylamine, used as the internal standard. With the proposed method, the contribution of the analyte to the global signal will be canceled, leading to the interference signal. It is possible to know whether an interference is present or not by working with the spectra at the top of the peak of the amphetamine. The procedure to calculate yref consists of finding its maximum and minimum possible values and plotting two spectra from these values. After this, a pair of wavelengths with the same absorbance values in both spectra must be found to apply eq 6. The minimum value of yref is found by plotting the hypothetical spectrum and increasing or decreasing its value until the absorbance at every wavelength is higher than zero. The minimum value of yref could 2562 Analytical Chemistry, Vol. 72, No. 11, June 1, 2000

Figure 3. Spectrum calculated for the interference by substituting y440 ) 0 into eq 4 (dashed line). The solid line represents the spectrum recorded at t ) 5 min. Inset: Plot of s440 - Kisi vs wavelength. s440 and si are the sample absorbances registered at the top of the amphetamine peak, at the reference wavelength, and at ith wavelength (the subscript i refers to the absorbance at a certain wavelength in the [1, j] range), respectively, and Ki is Ki,ref. For other details, see text.

not be found for amphetamine, however, since even considering yref ) 0 the values of the absorbances at every wavelength were positive. We have previously concluded19 that when yref ) 0 provides positive absorbances at every wavelength and when the values of sref - Ki,refsi at every wavelength are approximately null, there is no interference in the sample. Shown in Figure 3 is the spectrum calculated with yref ) 0 and the background spectrum from the baseline zone (at t ) 5 min). As it can be seen, they are quite similar. The inset in Figure 3 corresponds to the plot of sref - Ki,refsi vs the number of wavelengths measured. The values in this representation suggest the absence of interferences. The chromatographic analysis of amines was also carried out under normal-phase conditions. In this case, a sample containing a mixture of amphetamine and methamphetamine was injected. Figure 4 shows the chromatogram obtained after the injection of the sample (amphetamine tR ) 7.2 min and methamphetamine tR ) 7.6 min). Clearly, amphetamine and methamphetamine were not fully resolved, but we checked for the presence of additional compounds hidden in the peaks of both analytes. First, the spectra of the sample and of the analyte at the different positions of the peaks were obtained. Due to the features of the chromatographic system, the spectra were recorded every 4 nm. This is a long distance, taking into account the difficulty of finding wavelength pairs meeting the requirements established in the theoretical section. Therefore, we decided to calculate the absorbance at every nanometer by means of a cubic spline interpolation function. The spectra were also smoothed, using the Savitzky-Golay algorithm, because of the low values of the absorbances and their noisy appearance. The values of Ki,ref were calculated from the spectrum taken at the top of the peaks obtained after the injection of pure solutions

Figure 4. Chromatogram obtained at 275 nm after the injection of a sample containing 10 ppm of amphetamine (tR ) 7.2 min) and 10 ppm of methamphetamine (tR ) 7.6 min).

of amphetamine and methamphetamine. In our previous work,19 the reference wavelength was selected in such a way that the standard deviation of the sref - Ki,refsi values was minimum. In the present work, only the sample and two standards of amphetamine and methamphetamine were measured. The reference wavelength was arbitrarily selected as 440 nm. For the amphetamine peak, the interference spectra were calculated at t ) 7.07 and t ) 7.36 min. The minimum and maximum values of y440 at t ) 7.07 were 0.1 and 0.74 mAU, respectively. Several pairs of wavelengths meeting the requirement of no more than 5% difference in their absorbance values were found for both spectra, giving a prediction for the value of y440 in the range 0.11-0.57 mAU. By means of these values, the new maximum and minimum of the interference spectrum were calculated and the calculation of y440 was restarted: 15 wavelength pairs were found with their absorbance values coincidental within at least 95%. The final mean prediction of y440 and the standard deviation were 0.16 and 0.02, respectively. The process was developed in an analogous way to calculate the value of y440 at t ) 7.36 min. The first minimum-maximum range of y440 was 0.08-0.51, and after the first round of calculations, this range was reduced to 0.082-0.32. With this narrower range of values, the mean prediction and the standard deviation of y440 were 0.12 and 0.02, respectively (n ) 15). Figure 5 shows the spectra calculated at both times from these values and normalized to 1 in order to make possible their comparison. It can be seen that both spectra are almost coincidental. Their differences are probably due to the signal noise, since the absorbance values for both spectra were quite low (0.16 and 0.12 mAU, respectively, at the maximum of the absorption band of the spectra). In view of these results, we can conclude that, during the time interval 7.07-7.36 min, the same species (or mixture of species) is eluting simultaneously with amphetamine. The same procedure was followed for the determination of the spectra of the interference (amphetamine and whatever coelutes

Figure 5. Normalized spectra of the interference at t ) 7.07 min and t ) 7.36 min in the determination of amphetamine under normalphase conditions.

with it) at t ) 7.5 and t ) 7.77 min, considering methamphetamine as the analyte. The reference wavelength was set to 440 nm, and the Ki,ref values were calculated from the spectra obtained at the top of the chromatographic peak from a pure solution of methamphetamine. The final mean predictions (( the standard deviation) of the absorbances at y440 were 0.07 ( 0.02 and 0.06 ( 0.02 (n ) 16 for both cases) for the spectra at t ) 7.5 and t ) 7.77 min, respectively. The magnitudes of the signals were even lower than those obtained in the previous case, which explains why the standard deviation values were so high: 29% and 33%, respectively. These low signals could condition the goodness of the prediction of the interference spectrum, as can be seen in Figure 6, which shows the normalized spectra calculated by means of these values of y440 for the interference at both times. It can be seen that they are affected by the noise, but the shape of both spectra appear to be quite similar. The third case studied was the determination of bumetanide in the presence of ethacrynic acid (considered as an unknown and an unexpected compound in the sample). Figure 7 shows the chromatogram obtained after the injection of samples which contained both compounds. Only one peak was obtained, whose purity was investigated by means of the plots described previously. Several mixtures with the same concentration of ethacrynic acid but different known amounts of bumetanide were measured, always giving a single peak. The measurement of these mixtures permitted us to select the reference wavelength from the analyte spectrum by measuring the study of the standard deviation of the value of sref - Ki,refsi for every wavelength, as was described in ref 19 and indicated previously. Using 276 nm as the reference wavelength presented the maximum number of wavelengths having standard deviations of the sref - Ki,refsi values (n ) number of processed solutions for standard additions) under an arbitrarily fixed value (e.g., 276 nm as the reference wavelength provided 27 (sref - Ki,refsi) values with Analytical Chemistry, Vol. 72, No. 11, June 1, 2000

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Figure 6. Normalized spectra of the interference at t ) 7.5 min and t ) 7.77 min in the determination of methamphetamine under normal-phase conditions.

Figure 7. Chromatograms at 254 nm obtained for the samples of bumetanide and ethacrynic acid in concentrations of 50-100, 100100, 150-100, 200-100, and 250-100 ppm, respectively. Inset: Spectra of bumetanide (50 ppm, solid line) and ethacrynic acid (100 ppm, dashed line).

a standard deviation lower than 0.1, while 254 nm provided only 23 such values under the same conditions). The inset of Figure 8 shows the plot of the values of s276 Ki,refsi vs wavelength, where it can be seen that, in this case, the plot has its own features that are not reminescent of the background as in Figure 2. It is also shown that the spectrum calculated by entering y276 ) 0 in eq 4 presents negative values for some wavelengths. This clearly indicates that, in fact, the chromatographic peak was not pure. Calculation of the Concentration of the Analytes. For the determination of the concentration of the analytes in the sample, the spectrum of the interference at the top of the chromatographic 2564 Analytical Chemistry, Vol. 72, No. 11, June 1, 2000

Figure 8. Spectra calculated for ethacrynic acid in the determination of bumetanide (for a sample containing 50 ppm of bumetanide and 100 ppm of ethacrynic acid) by substituting y276 ) 0 (dashed line) and the maximum value, y276 ) s276 (solid line). Inset: Plot of s276 Kisi vs wavelength for all standard addition solutions prepared for the determination of bumetanide in the presence of ethacrynic acid. s276 and si are the sample absorbances registered at the top of the chromatographic peak, at the reference wavelength, and at ith wavelength (the subscript i refers to the absorbance at a certain wavelength in the [1, j] range), respectively, and Ki is Ki,ref. For other details, see text.

peak was calculated and then subtracted from the spectrum of the sample. This provided the analyte spectrum, which was then compared with the spectrum of a standard of known concentration. The procedure for the determination of the interference spectrum at the retention time of amphetamine (t ) 7.2 min) was analogous to that described for the peak purity investigation. Taking into account that the signals obtained are very low at 440 nm, and in view of Figure 5, the reference wavelength was set to 293 nm; the absorbance value at this wavelength is nearly the maximum, and the standard deviation of the predictions of this value was not expected to be as high as in the previous calculations. The calculations were started by fixing the minimum and maximum values of y293 (0.2-3.13); 0.2 mAU is the smallest absorbance value that provides non-negative absorbance values for the interference spectrum at any wavelength calculated from eq 4, and 3.13 mAU is the absorbance of the sample at t ) 7.2 min. This wide range was reduced after the first round of calculations to 0.2-1.84. With this new range, the mean prediction of yref was 0.63 mAU, having a standard deviation equal to 0.09 and n ) 19. The interference spectrum was calculated (see Figure 5) and subtracted from the sample spectrum to obtain the analyte spectrum, which was compared with the spectrum of a standard of 10 ppm of amphetamine; this yielded a prediction of 9.53 ppm of amphetemine in the sample, having an actual concentration of 10 ppm. The normalized calculated interference spectrum is displayed in the Figure 5, where it can be seen that it agrees with those calculated previously. The prediction of methamphetamine was carried out analogously, using 293 nm as reference wavelength. After a first round

of calculations, the minimum-maximum range was reduced, allowing the correct prediction of yref. The mean prediction of this value and its standard deviation were 0.50 mAU and 0.06 (n ) 27), respectively. In the Figure 6 can be seen the normalized spectrum calculated from the value of yref and by using eq 4. Despite the noise, the three spectra shown in Figure 6 are quite similar. The subtraction of the spectrum of the interference from the spectrum of the sample yielded to the spectrum of the analyte (methamphetamine), which was compared with the spectrum of a methamphetamine standard to determine the concentration of the sample. The concentration predicted for the methamphetamine in the sample was 10.07 ppm, while the real content was 10 ppm. It can be seen that accurate results were obtained. Two sets of mixtures with different amounts of ethacrynic acid were prepared for the determination of bumetanide (see Table 1). The procedure used to develop these calculations was the same as that described for the resolution of the amines, treating every sample individually and calculating the value of y276 for every one. The values found for y276 and the final determination of the bumetanide content in each sample can be found in the Table 1; the results obtained are quite satisfactory. Slight differences were found between the values of y276 for the solutions with the same concentration of ethacrynic acid, but these proved to be negligible, taking into account the magnitude of the signals; this was confirmed by the correct prediction of the concentration of bumetanide in all cases. CONCLUSIONS An adaptation of a method we have proposed19 for the spectrophotometric field to chromatography has been presented. Its usefulness has been demonstrated for three purposes: first, the detection of peak impurities hidden under a sample compound; second, the capability of predicting an interference spectrum at any position of a chromatographic peak; and third, the determi-

Table 1. Predictions of yref for Ethacrynic Acid and of the Content of Bumetanide in Coeluting Chromatographic Peaks predicted content (ppm) predicted bumetanide a bumetanide ethacrynic acid sref (mAU) yref (mAU) content (ppm) 50 100 50 50 50 50 100 100 100 100 100

50 100 150 200 50 100 150 200 250

5.7; 6 15.8; 16.2 44.1; 41.1 93.2; 92.2 126.8; 135.0 179.1; 165 52.3; 53.1 100.0; 100.0 141.3; 138.3 188.3; 196.3 225.2; 214.1

4.6 ( 0.3 5.6 ( 0.9 5.5 ( 0.7 5.1 ( 0.3 14.4 ( 0.6 15.0 ( 0.6 16 ( 1 16.4 ( 0.9 16 ( 1

44.8 99.6 142.82 198.2 44.62 98.18 143.91 198.48 241.23

a s ref is the absorbance at 274 nm for the retention time of the mixtures.

nation of an analyte in the presence of an unknown, and sometimes unexpected, interference. The HPCIM method presents advantages over the more traditional methods described in the introduction because it does not require the total separation of coeluting (if it is known that no more than two compounds are coeluting) species to calculate the spectrum of the interference, thereby avoiding optimization of the chromatographic conditions to resolve all the peaks. ACKNOWLEDGMENT We are grateful to the DGICYT (Project No. PB 97-1387) for financial support. Received for review June 15, 1999. Accepted January 20, 2000. AC990649Q

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