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Direct Quantitative Determination of Optically Active Absorbing Drugs in Human Urine by Circular Dichroism. Simultaneous Direct Determination of β-Ractam Antibiotics and Proteins
Direct Quantitative Determination of Optically Active Absorbing Drugs in Human Urine by Circular Dichroism. Simultaneous Direct Determination of p-Lactam Antibiotics and Proteins PlMR
G O R T ~ A MANUEL R ~ , RAVINA~~, AND JESUST.
VkQUEZtX
Received April 26, 1995, from the Xentro de Productos Naturales Organicos "Antonio Gunzalez", Universidad de La Laguna, Carretera de La Esperanza 2, 38206 La Laguna, Tenerife, Spain, and *Huspita/ Universitario de Canarias, Ofra, La Laguna, Tenerife, Spain. Accepted for publication August 7, 1995@.
Abstract 0 Over 200 urine samples from 61 subjects were analyzed by circular dichroism spectroscopy. The results proved that this technique can be applied to the direct determination of optically active absorbing drugs in urine of subjects under multiple drug administration, independently of the presence of proteins, which can simultaneously be determined. A list of noninterfering drugs is included. The validity of the present method was confirmed by analysis of variance, the plactam antibiotics ampicillin, cefoxitin, and cephalexin being chosen as model drugs and human albumin as the analytical standard for protein determination. The results demonstrated that the proposed method is accurate and precise, the correlation coefficients being higher than 0.9996. A circular dichroism and HPLC data comparison was successfully performed. The principal advantages of this method are simplicity, quickness, and economy, no derivatization or chromatographic separation steps being needed. Circular dichroism (CD) has become a routine technique for stereochemical studies, its most popular application being the determination of absolute configurations. Conformational analysis of low molecular weight organic compounds as well as macromolecules in solution, mainly proteins, is a second important application of this technique. In spite of the fact that its high degree of analytical selectivity allows an optically active absorbing compound in a mixture to be analyzed directly, as a result of the fact that two physical requirements (ellipticity and absorbance) are measured simultaneously, it is only recently that CD is being applied to chemical and pharmaceutical analysis.lS2 Moreover, the direct determination of optically active absorbing drugs in biological fluids has only been applied, a t present, to the direct determination of tris and tetrakis(hydroxyethy1)rutosides in urine and serum? and to tetracycline in urine.4 The present exhaustive study shows the applicability of CD to the direct quantitative determination of optically active absorbing drugs, exemplified with the direct determination of /3-lactam antibiotics in the voided urine of patients, excreting different amounts of proteins, under multiple drug administration. Ampicillin, cefoxitin, and cephalexin were chosen as model drugs because their CD spectral data are well e ~ t a b l i s h e d ,and ~ , ~they were part of the drug therapy of the patients under study. In addition, human albumin was chosen as the analytical standard for total urinary proteins determination and for CD spectral subtraction of the "complex" CD spectra, composed by the CD-active drug and proteins.
Experimental Section Materials-Human albumin (essentially fatty acid and globulin free), ampicillin sodium, amoxicillin, cefoxitin sodium, and cephalexin were obtained from Sigma Chemical Co. and used without further @Abstractpublished in Advance ACS Abstracts, September 15, 1995.
1316 / Journal of Pharmaceutical Sciences Vol. 84, No. 11, November 1995
purification. L-(+)-Ascorbic acid was obtained from Janssen Chimica. All drugs administered to the patients were required for their drug therapy. Subjects-Studies were performed on 61 volunteers: 10 healthy adult male subjects aged 20-25 years, and 51 patients, 38 males and 13 females, hospitalized in the urological service, aged 30-70 years. The 51 patients were randomly selected and receiving different pharmacology treatments. To carry out the present study, the corresponding ethical permission was obtained from the hospital committee, and informed consent was obtained from all participants. Blood analyses, including complete hemogram, and routine urinalyses were performed on each patient. Procedures-Specimen Collection-Between one and six urine samples were collected per subject, at different hours and/or on different days. For the hourly determination assays, the voided urine was collected and its volume measured every hour over a 6 h period, the collections being performed by means of a vesical catheter. These excreted urine samples corresponded to patients under the first administered dose. General Procedure for CD Analysis-All assays were carried out the same day of urine sample collection by diluting 20 pL of urine into a 5 mL calibrated flask with distilled water, namely, a 250 times dilution. Although this dilution can be changed, we have found that it offers the best chance of avoiding any possibility of interference from the urine metabolites. Standard Solutions of P-Lactam Antibiotics in Human UrineStandard solutions for each antibiotic were prepared by dissolving the accurately weighed compound in calibrated flasks with control human urine to obtain solutions containing 10 mg/mL. For concentrations in the range 1-20 pglmL, measured volumes of the standard solutions were diluted into calibrated flasks with human urine, and 20pL of the resulting solutions was diluted into 5 mL calibrated flasks with distilled water. For concentrations above 20 pglmL, measured volumes of the standard solutions were diluted into 5 mL calibrated flasks with distilled water. CD spectra of these solutions were recorded and the ellipticity angle a t the selected wavelength measured, in millidegrees (mdeg). Standard Solutions of Albumin in Human Urine-Standard solutions were prepared by dissolving the accurately weighed human albumin in calibrated flasks with control human urine to yield solutions containing 8 mg/mL. To obtain concentrations between 1 and 15 pglmL, measured volumes of the standard solutions were diluted into calibrated flasks with human urine, and 20 pL of the resulting solutions was diluted into 5 mL calibrated flasks with distilled water. For concentrations above 15 pg/mL, measured volumes of the standard solutions were diluted into 5 mL calibrated flasks with distilled water. CD spectra of these solutions were recorded, and the ellipticity angle at the selected wavelengths, 220 and 209 nm, were measured (mdeg). Standard Solutions of Cefoxitin-Albumin Mixtures in Human Urine-Standard solutions were prepared by dissolving the accurately weighed cefoxitin in calibrated flasks with control human urine to obtain solutions containing 10 mg/mL. To obtain concentrations between 1and 20 pg/mL, measured volumes of the standard solutions were diluted into calibrated flasks with human urine, and 20 pL of the resulting solutions was diluted into 5 mL calibrated flasks, each containing 48 p g h L of albumin, with distilled water. For concentrations above 20 pg/mL, measured volumes of the standard solutions were diluted into 5 mL calibrated flasks, each containing 48 pg/mL of albumin, with distilled water. Standard solutions for human albumin were prepared by dissolving the accurately weighed com-
0022-3549/953184-1316$09.00/0
0 1995, American Chemical Society and American Pharmaceutical Association
Table 1-Non-CD-Active Drugs in Urine Drug Class
234
Drug
Antimicrobial agents Hypnotics and sedatives Antidepressants Analgesics-antipyretics Non-steroidal anti-inflammatory agents Methylxanthine bronchodilators Coronary vasodilators Antihypertensive agents Antiemetics Antihistamines
Tobramycin, gentamycin, netilmycin, notfloxacin, pipemidic acid, ciprofloxacin, cotrimoxazole Alprazolam, triazolam Maprotiline hydrochloride Acetaminophen, amidopyrine Acetylsalicylic acid, diclofenac Theophylline Nitroglycerin, isosorbide dinitrate, nifedipine Prazosin Metoclopramide Astemizole, ranitidine
pound in calibrated flasks with distilled water to obtain solutions containing 8 mg/mL, and 30 pL was added t o the above mentioned calibrated flasks. CD spectra of these solutions were recorded and ellipticity angles measured (mdeg) (i) at 262 nm, to determine cefoxitin concentration, and (ii) a t 220 nm, to determine albumin concentration, upon CD spectral subtraction of the cefoxitin contribution. HPLC Analysis-The HPLC calibration and analysis of the p-lactam antibiotics was performed according to the method of Brisson and F ~ u r t i l l a n .Urine ~ samples were diluted with doubly distilled water, and 20 pL of the resulting solutions was injected into the liquid chromatograph without extraction. The mobile phase was a mixture of 0.01 M acetate buffer (pH 4.8) and methanol (85:15). Apparatus-CD spectra were obtained on a Jasco 5-600 spectropolarimeter (computer controlled) and recorded in the range 400-200 nm by using a 10 mm cylindrical quartz cell. All spectra were recorded at room temperature, with a band width of 1.0 nm and a step resolution of 0.1 nm. Generally, a sensitivity of 20 mdeg, a time constant of 1 s, a scan speed of 20 n d m i n , and one or two accumulations, were used. These last parameters were changed when a high or low concentration of the CD-active compound was detected or expected. Determinations from ellipticity angles having a strong photomultiplier HT voltage (above 460 V) are not accurate, dilution being recommended. Prior to measurements, the spectropolarimeter was calibrated with a standard solution of ammonium d-camphor10-sulfonate in distilled water. For HPLC measurements, a Shimadzu Corporation instrument was used, which included a liquid chromatography pump (model LC-7A) and a photodiode array UVvis detector (model SPD-M6A). A reversed-phase p-Bondapack (2-18 column (3.9 mm x 300 mm) was employed (Waters Assoc.).
__ ampicillin
207 206 -1 0
amoxicillin
.__._
Wavelength (nm)
200
300
Figure 1-Penicillins: CD spectrum of standard ampicillin (solid line) (13.30 pg/ mL) and of standard amoxicillin (dashed line) (7.39 pgimL).
~
....
223 -55 200
cefoxitin cephalexin
Wavelength (nm)
325
Figure 2-Cephalosporins: CD spectrum of standard cefoxitin (solid line) (40.53 pg/mL) and of standard cephalexin (dashed line) (19.79 pglmL). 15
Results and Discussion Non-CD-Active Urine Samples-The CD spectra of over 200 urine samples, collected from 10 healthy adult subjects and 51 hospitalized patients in the urological service, most of them under multiple drug administration and in many cases showing proteinuria, were analyzed. The CD spectra of the urine samples belonging to the healthy volunteers and to some patients under multiple drug administration, none of the drugs being optically active absorbing ones (non-CD-active), showed no Cotton effect in the 400-200 nm range, using a 250 times dilution. This expected result allowed us to perform the direct determination of an optically active absorbing drug in the presence of others that are non-CD-active. Table 1 shows different drugs that were administered to some of these patients and were not detected in their urine samples, a t the aforementioned dilution, as a consequence of the high degree of analytical selectivity of CD. Therefore the drugs shown in Table 1, in the range 400-200 nm, are nonabsorbing andor nonoptically active or possess an ellipticity too low to be detected at the analyzed concentration. CD-Active Urine Samples-The optically active absorbing drugs vitamin C, cephalexin, cefoxitin, ampicillin, or amoxicillin were detected in the CD spectra of the urine samples of the patients under treatment with one of the aforemen-
-10
vL--
200
I I
I
Wavelength (nm)
325
Figure 3 - 0 spectrum of standard vitamin C (20.02 pg/mL).
tioned drugs and, in many cases, simultaneously with others listed in Table 1. The pattern of these CD spectra changes, in the 200-250 nm range, depending on the presence of proteins, these with main absorptions below 250 nm.8 Thus, on the one hand, the CD spectra of those protein-free samples were superimposable with those of the standard solutions (Figures 1-3) after matching the concentrations. On the other hand, those samples containing, in addition to the CDactive drug, proteins exhibited CD spectra identical with those of the standard solutions in the range 400-250 nm, although Journal of Pharmaceutical Sciences / 1317 Vol. 84, No. 11, November 1995
35,
-35
1
200
I
I
-25
1
Wavelength (nm)
325
Figure 4-CD spectra of the urine of three patients under cefoxitin therapy (250 times dilutions): without proteins, solid line (23.25 &mL of cefoxitin);with proteins, dashed line (15.29pglmL of cefoxitin, and 11 .I9pg/mL of proteins) and dotted line (7.48pgimL of cefoxitin, and 17.16 pglmL of proteins). 5
-0 200
240
I
Wavelength (nm)
I
325
Figure 5 - 0 spectra of the urine of three patients under vitamin C therapy (250 times dilutions): without proteins, solid line (6.69 &mL of vitamin C); with proteins, dashed line (5.15 pglmL of vitamin C, and 2.14 pglmL of proteins) and dotted line (4.30 pg/mL of vitamin C, and 3.32 yglmL of proteins).
with modified patterns and intensities of the Cotton effects below 250 nm, due to the overlapping of the broad negative Cotton effect of the urinary proteins on those of the CD-active drugs. The complexity of these CD spectra depends on the ratio between the concentration of the CD-active drug and the urinary proteins. Furthermore, it will depend on the proximity of the wavelengths of the Cotton effects of the drug under analysis from the region of the proteins. Thus, only the first Cotton effect of the CD samples of some patients under cephalexin and cefoxitin therapies remained unaltered a t 255 and 262 nm, respectively, as can be observed for cefoxitin in Figure 4. As we will prove below, drugs with Cotton effects a t wavelengths above 250 nm can be straightforwardly determined by measuring the intensity of these Cotton effects, regardless of the presence of proteins. CD analysis of the urine samples of some patients under ampicillin, amoxicillin, and vitamin C therapies showed small bathochromic shifts of the wavelengths of their main Cotton effects, a t 234,235 and 248 nm, respectively, as a consequence of their location or close proximity to the protein region. Figure 5 shows this behavior for vitamin C. As we will discuss below, the simultaneous determination of these drugs and the total amount of proteins is feasible by spectral subtraction. As CD-active compounds, proteins can be analyzed by means of this technique. In fact, CD analysis of the urine 1318 / Journal of Pharmaceutical Sciences Vol. 84, No. 11, November 1995
200
Wavelength (nrn)
300
Figure 6-CD spectrum of standard human albumin (10.48pglmL).
samples of 31 patients which revealed in their routine urinalysis significant amounts of proteins (15-300 mg/dL) exhibited directly or after CD spectral subtraction of the CDactive drug clear protein-type CD spectra, namely, a broad negative Cotton effect with two extrema (around 220 and 209 nm) superimposable in most cases to that of human albumin (Figure 61, the main human urinary p r ~ t e i n . ~ Direct Determination of Optically Active Absorbing Drugs in Urine b y CD-p-Lactarn Antibiotics as Model Compounds-While the CD spectral patterns of penicillins are very similar: those of cephalosporins, including cephamycins, exhibit sufficient dissimilarities to discriminate among the cephalosporin homologues. In fact, we have recently classified these antibiotics in five CD spectroscopic groups on the basis of the wavelengths of their Cotton effects.6 Validation of the Method for /3-Lactam Antibiotics-To validate the present spectroscopic method, the p-lactam antibiotics cephalexin, cefoxitin and ampicillin were chosen as model drugs. The analysis of variance of the linear regression of the calibration line (ANOVA) carried out with these drugs confirms the linearity of the present method. The regression line equations of the linear relationship between the ellipticity angle and the concentration of the antibiotic were defined as 0 = mc z , where 0 is the ellipticity angle (mdeg) a t the selected wavelength, c is the concentration (ug/ mL), m is the slope of the fitted line, and z is the intercept of the regression line. Beer’s law is followed for concentrations up to 40 pg/mL for ampicillin, up to 80 pg/mL for cefoxitin, and up to 50 pg/mL for cephalexin, the correlation coefficient for each drug, r , being equal or higher than 0.9996. The root mean squares of the ANOVA error term (Soc) and the coefficient of variations (CV), obtained for these /3-lactam drugs demonstrate the validity of the method. Tables 2 and 3 summarize these results. The statistical analyses indicate that the obtained assay values were reproducible, with very satisfactory mean average coefficients of variations. On the basis of the precision and accuracy values shown in Table 3, the acceptable limit of quantitation is 5 pg/mL, the overall accuracies for ampicillin, cefoxitin, and cephalexin being 100.5,99.6, and 100.1%,with precisions of 1.69, 1.18, and 1.68%,respectively. In the present study the ratio of patients having proteinuria and those free of proteins favored the former and, therefore, “complex”CD spectra of optically active absorbing drugs were obtained more often than straightforward superimposable CD spectra. To study the feasibility of determining CD-active drugs from these “complex”CD spectra, human albumin was chosen as the protein standard for the determination of total urinary proteins, as well as to perform CD spectral subtrac-
+
Table 2-Parameters of Calibration Graphs for Model Drugs in Urine Compound
Range b g W
n
CD Lt inm)
Slope, m
Intercept, z
Ampicillin Cefoxitin Cephalexin
1.O-40.0 1.o-80.0 1.O-50.0
25 24 25
234 262 255
0.'9582 i0.0043 1.1500 0.0036 1.1766 i0.0067
-0.1769 0.0888 -0.2219 k 0.1671 -0.0717 k 0.2000
Correlation Coefficient, r
S,,
0.9998 0.9999 0.9996
0.288 0.534 0.634
+
*
Table 3-Data for Precision and Accuracy of Calibration Curve Standards of Model Drugs in Urine
Table 5-Data for Precision and Accuracy of Calibration Curve Standards of Human Albumin at Two Wavelengths (Aefi220 and 209 nm)
Concentration Mean Standard Mean of Calibration Determined Deviation Accuracy, Precision, Compound Standard, pg/mL n Value, pg/mL (SD) %of Nominal CV, %
Mean Mean Determined Standard Accuracy, Precision, Value, pg/mL Deviation (SD) % of Nominal CV, %
1.oo 5.00 10.00 20.00 40.00 1.oo 5.00 20.00 40.00 60.00 80.00
Ampicillin
Cefoxitin
1.oo
Cephalexin
5.00 10.00 20.00 40.00 50.00
5 5 5 5 5 5 4 3 3 4 5 5 4 3 3 5 5
0.99 5.13 10.04 19.80 40.06 1.09 5.00 19.59 40.35 59.73 80.12 1.07 5.12 9.94 19.83 39.50 50.44
0.032 0.079 0.298 0.284 0.333 0.063 0.118 0,111 0.452 0.414 0.626 0.016 0.114 0.253 0.273 0.260 0.808
99.0 102.6 100.4 99.0 100.1 109.0 100.0 98.0 100.9 99.6 100.1 107.0 102.4 99.4 99.2 98.8 100.9
3.23 1.54 2.97 1.43 0.83 5.78 2.36 0.57 1.12 0.69 0.78 1.49 2.23 2.54 1.38 0.66 1.60
Concentration of Calibration Standard,pg/rnL n 220 1.oo 5.00 15.00 30.00 50.00
209
220
5 0.99 0.99 0.050 5 4.88 4.99 0.094 5 15.12 15.10 0.094 5 30.17 29.84 0.258 5 49.85 50.08 0.335
209
220
209
0.120 99.0 99.0 0.230 98.0 99.8 0.131 100.8 100.6 0.404 100.6 99.5 0.800 99.7 100.2
220 209 5.05 12.1 1.93 4.60 0.62 0.87 0.85 1.35 0.67 1.60
110
0 (mdeg)
0
Table 4-Parameters of Calibration Graphs for Human Albumin in Urine Range lugimL)
CD
n
1.O-50.0 25 1.O-50.0 25
Lt(nm)Slope, m
220 209
Intercept, z
-1.5407 k 0.0046 4.3232 f 0.1229 -1.6319 f 0.0087 -0.3755 f 0.2325
Correlation Coefficient, r
So,
0.9999 0.9997
0.391 0.740 -1 30
200
tion. This protein was chosen due to the fact that it is the main urinary protein and that the CD spectra of those samples containing only proteins are almost identical with it. Validation of the Method for Albumin-The analysis of variance of the linear regression of the calibration line (ANOVA) carried out with human albumin a t 220 and 209 nm confirms the linearity of the method. The regression line equations of the linear relationship between the ellipticity angle a t 220 and 209 nm and the concentration of the albumin z. Beer's law is followed for were defined as 8 = mc concentrations up t o 50 pg/mL, the correlation coefficient, r, being equal o r higher than 0.9997 for each regression line equation. Table 4 summarizes these results. The statistical analyses indicate that the obtained assay values, by measuring at either 220 or 209 nm, were reproducible, and on the basis of the precision and accuracy values shown in Table 5, the acceptable limit of quantitation is 5 pg/ mL. Very satisfactory mean average coefficients of variations were obtained for both regression line equations, although the CV was slightly better for the one measured a t 220 nm. The determination of the total urinary proteins by CD spectroscopy was performed by assuming that the ellipticity of each urinary protein is identical with that of human albumin. The validity of this approach was confirmed by comparing our CD determinations, for the 31 patients with proteinuria, with those reported in their routine urinalysis performed in the hospital laboratory. Validation of the Method for P-Lactum Antibiotics-Albumin Mixtures in Human Urine-It is reasonable to think that a high concentration of urinary proteins could interfere in the
+
325
Wavelength (nm)
Figure 7-Complex CD spectra, from top to bottom, of in-house mixtures of albumin (48 pglmL) and cefoxitin (80, 60, 40, 20, 5, and 1 pg/mL), respectively. Table 6-Data for Precision and Accuracy of Calibration Curve Standard of Cefoxitin Applied to Cefoxitin-Albumin Mixtures Concentration Mean Standard Mean of Calibration Determined Deviation Accuracy, Precision, Compound Standard, pg/mL n Value, pg/mL (SD) % of Nominal CV, % Cefoxitin
1.00 5.00 20.00 40.00 60.00 80.00
6 6 6 6 6 6
0.74 4.80 20.06 40.57 60.86 81.64
0.097 0.086 0.175 0.230 0.230 0.459
74.0 96.0 100.3 101.4 101.4 102.0
13.11 1.79 0.87 0.57 0.38 0.56
detection and quantitation of these antibiotics. The overlapping of weak signals, from aromatic residues of proteins: and/ or extrinsic Cotton effects from drug-protein bindinglo could also be a problem a t wavelengths above 250 nm. To study how the presence of proteins can interfere with the determination of the P-lactam antibiotics, solutions having different concentrations of cefoxitin (1.0-80.0 pg/mL) and a constant concentration of albumin (48 pg/mL, equivalent to 1200 mg/ dL, a very high concentration of proteins in urine) were prepared and measured by CD (Figure 7). The concentration of cefoxitin was calculated by direct measurement at 262 nm and using the regression line equation for cefoxitin shown in Table 2. The results shown in Table 6 for cefoxitin, with overall accuracy and precision of 100.2% and 0.84% in the 5.0-80.0 pglmL range, respectively, were similar to those Journal of Pharmaceutical Sciences / 1319 Vol. 84, No. 11, November 1995
Table 7-Determinations of in-House Lactam-Albumin Mixtures
1 2 3 4 5 6
Compound
Drug
Albumin
Drug
Albumin
Cefoxitin
20.00 20.00 20.00 20.00 20.00 20.00
24.00 48.00 24.00 48.00 24.00 48.00
19.98 20.05 19.78 19.87 20.04 20.51
24.10 48.19 24.14 48.51 24.63 48.93
Cephalexin Ampicillin
234
Determined Concentration (Lcg/mL)
Prepared Concentration hglmL)
Entry
20
-20 200
Wavelength (nm)
300
Figure 10-Complex CD spectrum of the urine of a patient under ampicillin therapy (250 times dilution) (solid line), its calculated protein CD curve (dotted line) (2.54 pg/mL), and its calculated ampicillin CD spectrum (dashed line) (14.55 pg/mL).
-120
L
205
I 325
I
1
Wavelength (nm)
Figure 8-CD spectra of three in-house samples with a constant concentration of cephalexin (20 pg/mL) without albumin (solid line) and with 24 pg/mL (dashed line) and 48 pg/mL (dotted line) albumin. ”-
I
262
A
‘
-90 200
1
Wavelength (nm)
I
1
325
Figure 9-CD spectra of three in-house samples with a constant concentration of cefoxitin (20 pg/mL) without albumin (solid line) and with 24 pg/mL (dashed line) and 48 pg/mL (dotted line) albumin.
obtained in the study of cefoxitin in protein-free urine (Table 3) and confirmed the noninterference of proteins in the determinations of this type of antibiotics. This fact can easily be observed in Figures 8 and 9, where the intensity of the first Cotton effect at 255 and 262 nm, for cephalexin and cefoxitin, respectively, was independent of the concentration of proteins. See also Table 7 (entries 1-4). The above results also confirmed our previous statement that drugs with Cotton effects a t wavelengths above 250 nm can be straightforwardly determined, even in the presence of a high concentration of proteins, by measuring the intensity of these Cotton effects (Figures 4 and 7-9). The intensities of the Cotton effects, a t 209 and 220 nm, of albumin in the “complex”CD spectra shown in Figure 7 were 1320 / Journal of Pharmaceutical Sciences Vol. 84>No. 11, November 1995
measured directly for those samples having the two lower concentrations of cefoxitin. To measure the intensity of these Cotton effects, in the CD spectra of those samples having higher concentrations of cefoxitin, it was necessary to perform a computer subtraction of the drug’s spectra. Thus, these “complex”CD spectra became clear protein-type spectra, after removal of their corresponding CD drug contributions, determined as indicated above, their two main Cotton effects shifting to the correct Wavelengths. Following this procedure, the concentration of albumin (48.0 pg/mL) was determined by using the regression line equation shown in Table 4 (at 220 nm), an overall mean determined value of 48.15 pg/mL, a SD of 0.554, an overall accuracy of 100.3%,and a CV of 1.150%being obtained. These excellent data indicated that determination of the urinary proteins can be performed even when a high concentration of drug is present. In the case of drugs having the first Cotton effect a t wavelengths below 250 nm, the presence of proteins gives rise to “complex”CD spectra with shifted Cotton effects, as shown in Figure 5 for vitamin C. To determine both the concentrations of the drug and proteins in this type of spectra, it is necessary to perform a regression fit using the shapes of the drug and albumin spectra. In this way, the contributive drug and protein spectra of “complex” CD spectra were obtained. Figure 10 shows the “complex”CD spectrum (solid line) of a patient given ampicillin and its contributive standard and protein CD spectra (dashed and dotted lines, respectively). “he validity of this regression fit procedure, t o determine the concentration of the total urinary proteins and CD-active drugs with Cotton effects below 250 nm, can be observed for ampicillin in Table 7 (entries 5 and 6). Applications-To show the possibility of using CD spectroscopy in pharmacokinetic studies, the proposed method was applied to the hourly determination of ampicillin, cefoxitin, and cephalexin in the urine samples of nine patients, four of them with proteinuria, who were receiving intravenously the first dose of 1 g of one of these drugs together with other nonCD-active drugs (Table 1). The applicability of this method to monitor drugs can be observed in Figure 11, where a clear decrease in the CD spectra along the first 6 h period was obtained from the urine samples of a patient under cefoxitin therapy. The reported urinary excretion (%) in healthy volunteers for ampicillin, cefoxitin, and cephalexin are 90 k 8, 78, and 96, respectively.ll The cumulative (%) doses recovered in a 6 h period for our patients were 50, 63, and 76%for ampicillin; 59, 73, and 86% for cefoxitin; and 37, 46, and 64% for cephalexin. The lower values of the urinary excretion ob-
Conclusions The present CD study establishes a new analytical method for the direct quantitative determination of CD-active drugs in human urine, even in the presence of other non-CD-active drugs, and proteins, which can simultaneously be determined. Furthermore, the method is very economical, rapid, and simple, no derivatization or chromatographic separation steps being needed. In addition, the performed validation studies confirm the accuracy and precision of the proposed method. The properties of the present method are very practical for carrying out pharmacokinetic studies of optically active absorbing drugs.
-70I
200
325
Figure 11-CD spectra recorded during the 6 h period from the urine samples of a patient under cefoxitin therapy (250 times dilution) showing the gradual decrease in the intensity of their Cotton effects. From the first up to the sixth hour, the following concentrations of cefoxitin were determined: 53.54,37.68,25.11,21 .lo, 14.88, and 7.44 pg/mL. The total amounts of cefoxitin recovered in each hourly period were 349.4, 227.1, 113.4, 74.1, 59.7, and 39.3 mg, respectively. Table 8-Comparative Analysis by HPLC and CD Spectroscopy Determined Concentration (mg/mL) Entry
HPLC
Compound Ampicillin Cefoxitin Cephalexin
14.58 12.23 17.12 2.22 19.18 23.15 11.86 17.20 17.78
References and Notes
I
1
I
Wavelength (nm)
13.75 12.61 16.62 2.30 20.48 23.32 11.85 16.11 17.88
tained from some of these patients, compared with those reported in healthy volunteers, point to a renal insufficiency in these patients.12 To confirm these results a comparative analysis of nine random samples, one sample per patient, from the aforementioned hourly determination was carried out by means of CD and HPLC. Table 8 shows the excellent agreement between the values obtained by these techniques. The present comparative analysis confirms the validity of CD in performing a direct quantitative determination of CD-active drugs in human urine, independently of the presence of other non-CD-active drugs andor proteins. Currently, we are successfully performing the quantitative determination of this type of drug in protein-free serum, where concentration determinations will be very useful.
1. (a) Purdie, N.; Swallows, K. A. Anal. Chem. 1989,61,77A-89A. (b) Purdie, N. In Techniques and Instrumentation in Analytical Chemistry. Analytical Applications of Circular Dichroism; Purdie, N., Brittain, H. G., Eds.; Elsevier Science B. V.: Amsterdam, 1994; Vol. 14, pp 241-278. 2. Gergely, A. J. Pharm. Biomed. Anal. 1989, 7 , 523-541. 3. Jung, G.; Ottnad, M.; Voelter, W. Eur. J. Drug Metab. Pharmacokinet. 1977, 3, 131-141. 4. Bowen, J. M.; Purdie, N. J . Pharm. Sci. 1982, 71, 836-837. 5. Purdie, N.; Swallows, K. A. Anal. Chem. 1987,59, 1349-1351. 6. Gortazar, P.; Vazquez, J. T. J. Pharm. Sci. 1994,83,1204-1208. 7. Brisson, A. M.; Fourtillan, J. B. J. Chromatogr. 1981,223,393399. 8. Blout, E. R. In Fundamental Aspects and Recent Developments in Optical Rotatory Dispersion and Circular Dichroism; Ciardelli, F., Salvadori, P., Eds.; Heyden & Son L t d London, 1973; pp 352-372. 9. The presence of leukocytes and/or erythrocytes in the urine samples, besides proteins or optically active absorbing drugs, did not interfere with the CD analysis, but very noisy proteintype CD spectra were observed when the amount of proteins was very low and the concentration of blood cells was high (five cases). In these cases, a stronger UV absorption was observed in the same region where proteins absorb (below 250 nm), the extra absorption being tentatively assigned to the presence of cells. 10. Sjoholm, I.; Sjodin, T. Biochern. Pharm. 1972,21, 3041-3052. 11. Benet, L. Z.; Sheiner, L. B. In The Pharmacological Basis of Therapeutics; Gilman, A. G.; Goodman, L. S.; Gilman, A,, Eds.; Macmillan Publishing Co.: New York, 1980; pp 1675-1737. 12. Regamey, C.; Humair, L. Postgrad. Med. J . 1971, (Suppl., February), 69-78.
Acknowledgments Support of this work by the Direccion General de Investigacion Cientifica y TBcnica (DGICYT), Ministerio de Education y Ciencia (Spain), through grant PB93-0559 is gratefully acknowledged. We thank Prof. Julio D. Martin (University of La Laguna) for his valuable support and interest in this work. P. G. thanks the Excmo. Cabildo Insular de Tenerife (Spain) for a fellowship.
JS950174S
Journal of Pharmaceutical Sciences / 1321 Vol. 84, No. 11, November 1995
G O R T ~ A MANUEL R ~ , RAVINA~~, AND JESUST.
VkQUEZtX
Received April 26, 1995, from the Xentro de Productos Naturales Organicos "Antonio Gunzalez", Universidad de La Laguna, Carretera de La Esperanza 2, 38206 La Laguna, Tenerife, Spain, and *Huspita/ Universitario de Canarias, Ofra, La Laguna, Tenerife, Spain. Accepted for publication August 7, 1995@.
Abstract 0 Over 200 urine samples from 61 subjects were analyzed by circular dichroism spectroscopy. The results proved that this technique can be applied to the direct determination of optically active absorbing drugs in urine of subjects under multiple drug administration, independently of the presence of proteins, which can simultaneously be determined. A list of noninterfering drugs is included. The validity of the present method was confirmed by analysis of variance, the plactam antibiotics ampicillin, cefoxitin, and cephalexin being chosen as model drugs and human albumin as the analytical standard for protein determination. The results demonstrated that the proposed method is accurate and precise, the correlation coefficients being higher than 0.9996. A circular dichroism and HPLC data comparison was successfully performed. The principal advantages of this method are simplicity, quickness, and economy, no derivatization or chromatographic separation steps being needed. Circular dichroism (CD) has become a routine technique for stereochemical studies, its most popular application being the determination of absolute configurations. Conformational analysis of low molecular weight organic compounds as well as macromolecules in solution, mainly proteins, is a second important application of this technique. In spite of the fact that its high degree of analytical selectivity allows an optically active absorbing compound in a mixture to be analyzed directly, as a result of the fact that two physical requirements (ellipticity and absorbance) are measured simultaneously, it is only recently that CD is being applied to chemical and pharmaceutical analysis.lS2 Moreover, the direct determination of optically active absorbing drugs in biological fluids has only been applied, a t present, to the direct determination of tris and tetrakis(hydroxyethy1)rutosides in urine and serum? and to tetracycline in urine.4 The present exhaustive study shows the applicability of CD to the direct quantitative determination of optically active absorbing drugs, exemplified with the direct determination of /3-lactam antibiotics in the voided urine of patients, excreting different amounts of proteins, under multiple drug administration. Ampicillin, cefoxitin, and cephalexin were chosen as model drugs because their CD spectral data are well e ~ t a b l i s h e d ,and ~ , ~they were part of the drug therapy of the patients under study. In addition, human albumin was chosen as the analytical standard for total urinary proteins determination and for CD spectral subtraction of the "complex" CD spectra, composed by the CD-active drug and proteins.
Experimental Section Materials-Human albumin (essentially fatty acid and globulin free), ampicillin sodium, amoxicillin, cefoxitin sodium, and cephalexin were obtained from Sigma Chemical Co. and used without further @Abstractpublished in Advance ACS Abstracts, September 15, 1995.
1316 / Journal of Pharmaceutical Sciences Vol. 84, No. 11, November 1995
purification. L-(+)-Ascorbic acid was obtained from Janssen Chimica. All drugs administered to the patients were required for their drug therapy. Subjects-Studies were performed on 61 volunteers: 10 healthy adult male subjects aged 20-25 years, and 51 patients, 38 males and 13 females, hospitalized in the urological service, aged 30-70 years. The 51 patients were randomly selected and receiving different pharmacology treatments. To carry out the present study, the corresponding ethical permission was obtained from the hospital committee, and informed consent was obtained from all participants. Blood analyses, including complete hemogram, and routine urinalyses were performed on each patient. Procedures-Specimen Collection-Between one and six urine samples were collected per subject, at different hours and/or on different days. For the hourly determination assays, the voided urine was collected and its volume measured every hour over a 6 h period, the collections being performed by means of a vesical catheter. These excreted urine samples corresponded to patients under the first administered dose. General Procedure for CD Analysis-All assays were carried out the same day of urine sample collection by diluting 20 pL of urine into a 5 mL calibrated flask with distilled water, namely, a 250 times dilution. Although this dilution can be changed, we have found that it offers the best chance of avoiding any possibility of interference from the urine metabolites. Standard Solutions of P-Lactam Antibiotics in Human UrineStandard solutions for each antibiotic were prepared by dissolving the accurately weighed compound in calibrated flasks with control human urine to obtain solutions containing 10 mg/mL. For concentrations in the range 1-20 pglmL, measured volumes of the standard solutions were diluted into calibrated flasks with human urine, and 20pL of the resulting solutions was diluted into 5 mL calibrated flasks with distilled water. For concentrations above 20 pglmL, measured volumes of the standard solutions were diluted into 5 mL calibrated flasks with distilled water. CD spectra of these solutions were recorded and the ellipticity angle a t the selected wavelength measured, in millidegrees (mdeg). Standard Solutions of Albumin in Human Urine-Standard solutions were prepared by dissolving the accurately weighed human albumin in calibrated flasks with control human urine to yield solutions containing 8 mg/mL. To obtain concentrations between 1 and 15 pglmL, measured volumes of the standard solutions were diluted into calibrated flasks with human urine, and 20 pL of the resulting solutions was diluted into 5 mL calibrated flasks with distilled water. For concentrations above 15 pg/mL, measured volumes of the standard solutions were diluted into 5 mL calibrated flasks with distilled water. CD spectra of these solutions were recorded, and the ellipticity angle at the selected wavelengths, 220 and 209 nm, were measured (mdeg). Standard Solutions of Cefoxitin-Albumin Mixtures in Human Urine-Standard solutions were prepared by dissolving the accurately weighed cefoxitin in calibrated flasks with control human urine to obtain solutions containing 10 mg/mL. To obtain concentrations between 1and 20 pg/mL, measured volumes of the standard solutions were diluted into calibrated flasks with human urine, and 20 pL of the resulting solutions was diluted into 5 mL calibrated flasks, each containing 48 p g h L of albumin, with distilled water. For concentrations above 20 pg/mL, measured volumes of the standard solutions were diluted into 5 mL calibrated flasks, each containing 48 pg/mL of albumin, with distilled water. Standard solutions for human albumin were prepared by dissolving the accurately weighed com-
0022-3549/953184-1316$09.00/0
0 1995, American Chemical Society and American Pharmaceutical Association
Table 1-Non-CD-Active Drugs in Urine Drug Class
234
Drug
Antimicrobial agents Hypnotics and sedatives Antidepressants Analgesics-antipyretics Non-steroidal anti-inflammatory agents Methylxanthine bronchodilators Coronary vasodilators Antihypertensive agents Antiemetics Antihistamines
Tobramycin, gentamycin, netilmycin, notfloxacin, pipemidic acid, ciprofloxacin, cotrimoxazole Alprazolam, triazolam Maprotiline hydrochloride Acetaminophen, amidopyrine Acetylsalicylic acid, diclofenac Theophylline Nitroglycerin, isosorbide dinitrate, nifedipine Prazosin Metoclopramide Astemizole, ranitidine
pound in calibrated flasks with distilled water to obtain solutions containing 8 mg/mL, and 30 pL was added t o the above mentioned calibrated flasks. CD spectra of these solutions were recorded and ellipticity angles measured (mdeg) (i) at 262 nm, to determine cefoxitin concentration, and (ii) a t 220 nm, to determine albumin concentration, upon CD spectral subtraction of the cefoxitin contribution. HPLC Analysis-The HPLC calibration and analysis of the p-lactam antibiotics was performed according to the method of Brisson and F ~ u r t i l l a n .Urine ~ samples were diluted with doubly distilled water, and 20 pL of the resulting solutions was injected into the liquid chromatograph without extraction. The mobile phase was a mixture of 0.01 M acetate buffer (pH 4.8) and methanol (85:15). Apparatus-CD spectra were obtained on a Jasco 5-600 spectropolarimeter (computer controlled) and recorded in the range 400-200 nm by using a 10 mm cylindrical quartz cell. All spectra were recorded at room temperature, with a band width of 1.0 nm and a step resolution of 0.1 nm. Generally, a sensitivity of 20 mdeg, a time constant of 1 s, a scan speed of 20 n d m i n , and one or two accumulations, were used. These last parameters were changed when a high or low concentration of the CD-active compound was detected or expected. Determinations from ellipticity angles having a strong photomultiplier HT voltage (above 460 V) are not accurate, dilution being recommended. Prior to measurements, the spectropolarimeter was calibrated with a standard solution of ammonium d-camphor10-sulfonate in distilled water. For HPLC measurements, a Shimadzu Corporation instrument was used, which included a liquid chromatography pump (model LC-7A) and a photodiode array UVvis detector (model SPD-M6A). A reversed-phase p-Bondapack (2-18 column (3.9 mm x 300 mm) was employed (Waters Assoc.).
__ ampicillin
207 206 -1 0
amoxicillin
.__._
Wavelength (nm)
200
300
Figure 1-Penicillins: CD spectrum of standard ampicillin (solid line) (13.30 pg/ mL) and of standard amoxicillin (dashed line) (7.39 pgimL).
~
....
223 -55 200
cefoxitin cephalexin
Wavelength (nm)
325
Figure 2-Cephalosporins: CD spectrum of standard cefoxitin (solid line) (40.53 pg/mL) and of standard cephalexin (dashed line) (19.79 pglmL). 15
Results and Discussion Non-CD-Active Urine Samples-The CD spectra of over 200 urine samples, collected from 10 healthy adult subjects and 51 hospitalized patients in the urological service, most of them under multiple drug administration and in many cases showing proteinuria, were analyzed. The CD spectra of the urine samples belonging to the healthy volunteers and to some patients under multiple drug administration, none of the drugs being optically active absorbing ones (non-CD-active), showed no Cotton effect in the 400-200 nm range, using a 250 times dilution. This expected result allowed us to perform the direct determination of an optically active absorbing drug in the presence of others that are non-CD-active. Table 1 shows different drugs that were administered to some of these patients and were not detected in their urine samples, a t the aforementioned dilution, as a consequence of the high degree of analytical selectivity of CD. Therefore the drugs shown in Table 1, in the range 400-200 nm, are nonabsorbing andor nonoptically active or possess an ellipticity too low to be detected at the analyzed concentration. CD-Active Urine Samples-The optically active absorbing drugs vitamin C, cephalexin, cefoxitin, ampicillin, or amoxicillin were detected in the CD spectra of the urine samples of the patients under treatment with one of the aforemen-
-10
vL--
200
I I
I
Wavelength (nm)
325
Figure 3 - 0 spectrum of standard vitamin C (20.02 pg/mL).
tioned drugs and, in many cases, simultaneously with others listed in Table 1. The pattern of these CD spectra changes, in the 200-250 nm range, depending on the presence of proteins, these with main absorptions below 250 nm.8 Thus, on the one hand, the CD spectra of those protein-free samples were superimposable with those of the standard solutions (Figures 1-3) after matching the concentrations. On the other hand, those samples containing, in addition to the CDactive drug, proteins exhibited CD spectra identical with those of the standard solutions in the range 400-250 nm, although Journal of Pharmaceutical Sciences / 1317 Vol. 84, No. 11, November 1995
35,
-35
1
200
I
I
-25
1
Wavelength (nm)
325
Figure 4-CD spectra of the urine of three patients under cefoxitin therapy (250 times dilutions): without proteins, solid line (23.25 &mL of cefoxitin);with proteins, dashed line (15.29pglmL of cefoxitin, and 11 .I9pg/mL of proteins) and dotted line (7.48pgimL of cefoxitin, and 17.16 pglmL of proteins). 5
-0 200
240
I
Wavelength (nm)
I
325
Figure 5 - 0 spectra of the urine of three patients under vitamin C therapy (250 times dilutions): without proteins, solid line (6.69 &mL of vitamin C); with proteins, dashed line (5.15 pglmL of vitamin C, and 2.14 pglmL of proteins) and dotted line (4.30 pg/mL of vitamin C, and 3.32 yglmL of proteins).
with modified patterns and intensities of the Cotton effects below 250 nm, due to the overlapping of the broad negative Cotton effect of the urinary proteins on those of the CD-active drugs. The complexity of these CD spectra depends on the ratio between the concentration of the CD-active drug and the urinary proteins. Furthermore, it will depend on the proximity of the wavelengths of the Cotton effects of the drug under analysis from the region of the proteins. Thus, only the first Cotton effect of the CD samples of some patients under cephalexin and cefoxitin therapies remained unaltered a t 255 and 262 nm, respectively, as can be observed for cefoxitin in Figure 4. As we will prove below, drugs with Cotton effects a t wavelengths above 250 nm can be straightforwardly determined by measuring the intensity of these Cotton effects, regardless of the presence of proteins. CD analysis of the urine samples of some patients under ampicillin, amoxicillin, and vitamin C therapies showed small bathochromic shifts of the wavelengths of their main Cotton effects, a t 234,235 and 248 nm, respectively, as a consequence of their location or close proximity to the protein region. Figure 5 shows this behavior for vitamin C. As we will discuss below, the simultaneous determination of these drugs and the total amount of proteins is feasible by spectral subtraction. As CD-active compounds, proteins can be analyzed by means of this technique. In fact, CD analysis of the urine 1318 / Journal of Pharmaceutical Sciences Vol. 84, No. 11, November 1995
200
Wavelength (nrn)
300
Figure 6-CD spectrum of standard human albumin (10.48pglmL).
samples of 31 patients which revealed in their routine urinalysis significant amounts of proteins (15-300 mg/dL) exhibited directly or after CD spectral subtraction of the CDactive drug clear protein-type CD spectra, namely, a broad negative Cotton effect with two extrema (around 220 and 209 nm) superimposable in most cases to that of human albumin (Figure 61, the main human urinary p r ~ t e i n . ~ Direct Determination of Optically Active Absorbing Drugs in Urine b y CD-p-Lactarn Antibiotics as Model Compounds-While the CD spectral patterns of penicillins are very similar: those of cephalosporins, including cephamycins, exhibit sufficient dissimilarities to discriminate among the cephalosporin homologues. In fact, we have recently classified these antibiotics in five CD spectroscopic groups on the basis of the wavelengths of their Cotton effects.6 Validation of the Method for /3-Lactam Antibiotics-To validate the present spectroscopic method, the p-lactam antibiotics cephalexin, cefoxitin and ampicillin were chosen as model drugs. The analysis of variance of the linear regression of the calibration line (ANOVA) carried out with these drugs confirms the linearity of the present method. The regression line equations of the linear relationship between the ellipticity angle and the concentration of the antibiotic were defined as 0 = mc z , where 0 is the ellipticity angle (mdeg) a t the selected wavelength, c is the concentration (ug/ mL), m is the slope of the fitted line, and z is the intercept of the regression line. Beer’s law is followed for concentrations up to 40 pg/mL for ampicillin, up to 80 pg/mL for cefoxitin, and up to 50 pg/mL for cephalexin, the correlation coefficient for each drug, r , being equal or higher than 0.9996. The root mean squares of the ANOVA error term (Soc) and the coefficient of variations (CV), obtained for these /3-lactam drugs demonstrate the validity of the method. Tables 2 and 3 summarize these results. The statistical analyses indicate that the obtained assay values were reproducible, with very satisfactory mean average coefficients of variations. On the basis of the precision and accuracy values shown in Table 3, the acceptable limit of quantitation is 5 pg/mL, the overall accuracies for ampicillin, cefoxitin, and cephalexin being 100.5,99.6, and 100.1%,with precisions of 1.69, 1.18, and 1.68%,respectively. In the present study the ratio of patients having proteinuria and those free of proteins favored the former and, therefore, “complex”CD spectra of optically active absorbing drugs were obtained more often than straightforward superimposable CD spectra. To study the feasibility of determining CD-active drugs from these “complex”CD spectra, human albumin was chosen as the protein standard for the determination of total urinary proteins, as well as to perform CD spectral subtrac-
+
Table 2-Parameters of Calibration Graphs for Model Drugs in Urine Compound
Range b g W
n
CD Lt inm)
Slope, m
Intercept, z
Ampicillin Cefoxitin Cephalexin
1.O-40.0 1.o-80.0 1.O-50.0
25 24 25
234 262 255
0.'9582 i0.0043 1.1500 0.0036 1.1766 i0.0067
-0.1769 0.0888 -0.2219 k 0.1671 -0.0717 k 0.2000
Correlation Coefficient, r
S,,
0.9998 0.9999 0.9996
0.288 0.534 0.634
+
*
Table 3-Data for Precision and Accuracy of Calibration Curve Standards of Model Drugs in Urine
Table 5-Data for Precision and Accuracy of Calibration Curve Standards of Human Albumin at Two Wavelengths (Aefi220 and 209 nm)
Concentration Mean Standard Mean of Calibration Determined Deviation Accuracy, Precision, Compound Standard, pg/mL n Value, pg/mL (SD) %of Nominal CV, %
Mean Mean Determined Standard Accuracy, Precision, Value, pg/mL Deviation (SD) % of Nominal CV, %
1.oo 5.00 10.00 20.00 40.00 1.oo 5.00 20.00 40.00 60.00 80.00
Ampicillin
Cefoxitin
1.oo
Cephalexin
5.00 10.00 20.00 40.00 50.00
5 5 5 5 5 5 4 3 3 4 5 5 4 3 3 5 5
0.99 5.13 10.04 19.80 40.06 1.09 5.00 19.59 40.35 59.73 80.12 1.07 5.12 9.94 19.83 39.50 50.44
0.032 0.079 0.298 0.284 0.333 0.063 0.118 0,111 0.452 0.414 0.626 0.016 0.114 0.253 0.273 0.260 0.808
99.0 102.6 100.4 99.0 100.1 109.0 100.0 98.0 100.9 99.6 100.1 107.0 102.4 99.4 99.2 98.8 100.9
3.23 1.54 2.97 1.43 0.83 5.78 2.36 0.57 1.12 0.69 0.78 1.49 2.23 2.54 1.38 0.66 1.60
Concentration of Calibration Standard,pg/rnL n 220 1.oo 5.00 15.00 30.00 50.00
209
220
5 0.99 0.99 0.050 5 4.88 4.99 0.094 5 15.12 15.10 0.094 5 30.17 29.84 0.258 5 49.85 50.08 0.335
209
220
209
0.120 99.0 99.0 0.230 98.0 99.8 0.131 100.8 100.6 0.404 100.6 99.5 0.800 99.7 100.2
220 209 5.05 12.1 1.93 4.60 0.62 0.87 0.85 1.35 0.67 1.60
110
0 (mdeg)
0
Table 4-Parameters of Calibration Graphs for Human Albumin in Urine Range lugimL)
CD
n
1.O-50.0 25 1.O-50.0 25
Lt(nm)Slope, m
220 209
Intercept, z
-1.5407 k 0.0046 4.3232 f 0.1229 -1.6319 f 0.0087 -0.3755 f 0.2325
Correlation Coefficient, r
So,
0.9999 0.9997
0.391 0.740 -1 30
200
tion. This protein was chosen due to the fact that it is the main urinary protein and that the CD spectra of those samples containing only proteins are almost identical with it. Validation of the Method for Albumin-The analysis of variance of the linear regression of the calibration line (ANOVA) carried out with human albumin a t 220 and 209 nm confirms the linearity of the method. The regression line equations of the linear relationship between the ellipticity angle a t 220 and 209 nm and the concentration of the albumin z. Beer's law is followed for were defined as 8 = mc concentrations up t o 50 pg/mL, the correlation coefficient, r, being equal o r higher than 0.9997 for each regression line equation. Table 4 summarizes these results. The statistical analyses indicate that the obtained assay values, by measuring at either 220 or 209 nm, were reproducible, and on the basis of the precision and accuracy values shown in Table 5, the acceptable limit of quantitation is 5 pg/ mL. Very satisfactory mean average coefficients of variations were obtained for both regression line equations, although the CV was slightly better for the one measured a t 220 nm. The determination of the total urinary proteins by CD spectroscopy was performed by assuming that the ellipticity of each urinary protein is identical with that of human albumin. The validity of this approach was confirmed by comparing our CD determinations, for the 31 patients with proteinuria, with those reported in their routine urinalysis performed in the hospital laboratory. Validation of the Method for P-Lactum Antibiotics-Albumin Mixtures in Human Urine-It is reasonable to think that a high concentration of urinary proteins could interfere in the
+
325
Wavelength (nm)
Figure 7-Complex CD spectra, from top to bottom, of in-house mixtures of albumin (48 pglmL) and cefoxitin (80, 60, 40, 20, 5, and 1 pg/mL), respectively. Table 6-Data for Precision and Accuracy of Calibration Curve Standard of Cefoxitin Applied to Cefoxitin-Albumin Mixtures Concentration Mean Standard Mean of Calibration Determined Deviation Accuracy, Precision, Compound Standard, pg/mL n Value, pg/mL (SD) % of Nominal CV, % Cefoxitin
1.00 5.00 20.00 40.00 60.00 80.00
6 6 6 6 6 6
0.74 4.80 20.06 40.57 60.86 81.64
0.097 0.086 0.175 0.230 0.230 0.459
74.0 96.0 100.3 101.4 101.4 102.0
13.11 1.79 0.87 0.57 0.38 0.56
detection and quantitation of these antibiotics. The overlapping of weak signals, from aromatic residues of proteins: and/ or extrinsic Cotton effects from drug-protein bindinglo could also be a problem a t wavelengths above 250 nm. To study how the presence of proteins can interfere with the determination of the P-lactam antibiotics, solutions having different concentrations of cefoxitin (1.0-80.0 pg/mL) and a constant concentration of albumin (48 pg/mL, equivalent to 1200 mg/ dL, a very high concentration of proteins in urine) were prepared and measured by CD (Figure 7). The concentration of cefoxitin was calculated by direct measurement at 262 nm and using the regression line equation for cefoxitin shown in Table 2. The results shown in Table 6 for cefoxitin, with overall accuracy and precision of 100.2% and 0.84% in the 5.0-80.0 pglmL range, respectively, were similar to those Journal of Pharmaceutical Sciences / 1319 Vol. 84, No. 11, November 1995
Table 7-Determinations of in-House Lactam-Albumin Mixtures
1 2 3 4 5 6
Compound
Drug
Albumin
Drug
Albumin
Cefoxitin
20.00 20.00 20.00 20.00 20.00 20.00
24.00 48.00 24.00 48.00 24.00 48.00
19.98 20.05 19.78 19.87 20.04 20.51
24.10 48.19 24.14 48.51 24.63 48.93
Cephalexin Ampicillin
234
Determined Concentration (Lcg/mL)
Prepared Concentration hglmL)
Entry
20
-20 200
Wavelength (nm)
300
Figure 10-Complex CD spectrum of the urine of a patient under ampicillin therapy (250 times dilution) (solid line), its calculated protein CD curve (dotted line) (2.54 pg/mL), and its calculated ampicillin CD spectrum (dashed line) (14.55 pg/mL).
-120
L
205
I 325
I
1
Wavelength (nm)
Figure 8-CD spectra of three in-house samples with a constant concentration of cephalexin (20 pg/mL) without albumin (solid line) and with 24 pg/mL (dashed line) and 48 pg/mL (dotted line) albumin. ”-
I
262
A
‘
-90 200
1
Wavelength (nm)
I
1
325
Figure 9-CD spectra of three in-house samples with a constant concentration of cefoxitin (20 pg/mL) without albumin (solid line) and with 24 pg/mL (dashed line) and 48 pg/mL (dotted line) albumin.
obtained in the study of cefoxitin in protein-free urine (Table 3) and confirmed the noninterference of proteins in the determinations of this type of antibiotics. This fact can easily be observed in Figures 8 and 9, where the intensity of the first Cotton effect at 255 and 262 nm, for cephalexin and cefoxitin, respectively, was independent of the concentration of proteins. See also Table 7 (entries 1-4). The above results also confirmed our previous statement that drugs with Cotton effects a t wavelengths above 250 nm can be straightforwardly determined, even in the presence of a high concentration of proteins, by measuring the intensity of these Cotton effects (Figures 4 and 7-9). The intensities of the Cotton effects, a t 209 and 220 nm, of albumin in the “complex”CD spectra shown in Figure 7 were 1320 / Journal of Pharmaceutical Sciences Vol. 84>No. 11, November 1995
measured directly for those samples having the two lower concentrations of cefoxitin. To measure the intensity of these Cotton effects, in the CD spectra of those samples having higher concentrations of cefoxitin, it was necessary to perform a computer subtraction of the drug’s spectra. Thus, these “complex”CD spectra became clear protein-type spectra, after removal of their corresponding CD drug contributions, determined as indicated above, their two main Cotton effects shifting to the correct Wavelengths. Following this procedure, the concentration of albumin (48.0 pg/mL) was determined by using the regression line equation shown in Table 4 (at 220 nm), an overall mean determined value of 48.15 pg/mL, a SD of 0.554, an overall accuracy of 100.3%,and a CV of 1.150%being obtained. These excellent data indicated that determination of the urinary proteins can be performed even when a high concentration of drug is present. In the case of drugs having the first Cotton effect a t wavelengths below 250 nm, the presence of proteins gives rise to “complex”CD spectra with shifted Cotton effects, as shown in Figure 5 for vitamin C. To determine both the concentrations of the drug and proteins in this type of spectra, it is necessary to perform a regression fit using the shapes of the drug and albumin spectra. In this way, the contributive drug and protein spectra of “complex” CD spectra were obtained. Figure 10 shows the “complex”CD spectrum (solid line) of a patient given ampicillin and its contributive standard and protein CD spectra (dashed and dotted lines, respectively). “he validity of this regression fit procedure, t o determine the concentration of the total urinary proteins and CD-active drugs with Cotton effects below 250 nm, can be observed for ampicillin in Table 7 (entries 5 and 6). Applications-To show the possibility of using CD spectroscopy in pharmacokinetic studies, the proposed method was applied to the hourly determination of ampicillin, cefoxitin, and cephalexin in the urine samples of nine patients, four of them with proteinuria, who were receiving intravenously the first dose of 1 g of one of these drugs together with other nonCD-active drugs (Table 1). The applicability of this method to monitor drugs can be observed in Figure 11, where a clear decrease in the CD spectra along the first 6 h period was obtained from the urine samples of a patient under cefoxitin therapy. The reported urinary excretion (%) in healthy volunteers for ampicillin, cefoxitin, and cephalexin are 90 k 8, 78, and 96, respectively.ll The cumulative (%) doses recovered in a 6 h period for our patients were 50, 63, and 76%for ampicillin; 59, 73, and 86% for cefoxitin; and 37, 46, and 64% for cephalexin. The lower values of the urinary excretion ob-
Conclusions The present CD study establishes a new analytical method for the direct quantitative determination of CD-active drugs in human urine, even in the presence of other non-CD-active drugs, and proteins, which can simultaneously be determined. Furthermore, the method is very economical, rapid, and simple, no derivatization or chromatographic separation steps being needed. In addition, the performed validation studies confirm the accuracy and precision of the proposed method. The properties of the present method are very practical for carrying out pharmacokinetic studies of optically active absorbing drugs.
-70I
200
325
Figure 11-CD spectra recorded during the 6 h period from the urine samples of a patient under cefoxitin therapy (250 times dilution) showing the gradual decrease in the intensity of their Cotton effects. From the first up to the sixth hour, the following concentrations of cefoxitin were determined: 53.54,37.68,25.11,21 .lo, 14.88, and 7.44 pg/mL. The total amounts of cefoxitin recovered in each hourly period were 349.4, 227.1, 113.4, 74.1, 59.7, and 39.3 mg, respectively. Table 8-Comparative Analysis by HPLC and CD Spectroscopy Determined Concentration (mg/mL) Entry
HPLC
Compound Ampicillin Cefoxitin Cephalexin
14.58 12.23 17.12 2.22 19.18 23.15 11.86 17.20 17.78
References and Notes
I
1
I
Wavelength (nm)
13.75 12.61 16.62 2.30 20.48 23.32 11.85 16.11 17.88
tained from some of these patients, compared with those reported in healthy volunteers, point to a renal insufficiency in these patients.12 To confirm these results a comparative analysis of nine random samples, one sample per patient, from the aforementioned hourly determination was carried out by means of CD and HPLC. Table 8 shows the excellent agreement between the values obtained by these techniques. The present comparative analysis confirms the validity of CD in performing a direct quantitative determination of CD-active drugs in human urine, independently of the presence of other non-CD-active drugs andor proteins. Currently, we are successfully performing the quantitative determination of this type of drug in protein-free serum, where concentration determinations will be very useful.
1. (a) Purdie, N.; Swallows, K. A. Anal. Chem. 1989,61,77A-89A. (b) Purdie, N. In Techniques and Instrumentation in Analytical Chemistry. Analytical Applications of Circular Dichroism; Purdie, N., Brittain, H. G., Eds.; Elsevier Science B. V.: Amsterdam, 1994; Vol. 14, pp 241-278. 2. Gergely, A. J. Pharm. Biomed. Anal. 1989, 7 , 523-541. 3. Jung, G.; Ottnad, M.; Voelter, W. Eur. J. Drug Metab. Pharmacokinet. 1977, 3, 131-141. 4. Bowen, J. M.; Purdie, N. J . Pharm. Sci. 1982, 71, 836-837. 5. Purdie, N.; Swallows, K. A. Anal. Chem. 1987,59, 1349-1351. 6. Gortazar, P.; Vazquez, J. T. J. Pharm. Sci. 1994,83,1204-1208. 7. Brisson, A. M.; Fourtillan, J. B. J. Chromatogr. 1981,223,393399. 8. Blout, E. R. In Fundamental Aspects and Recent Developments in Optical Rotatory Dispersion and Circular Dichroism; Ciardelli, F., Salvadori, P., Eds.; Heyden & Son L t d London, 1973; pp 352-372. 9. The presence of leukocytes and/or erythrocytes in the urine samples, besides proteins or optically active absorbing drugs, did not interfere with the CD analysis, but very noisy proteintype CD spectra were observed when the amount of proteins was very low and the concentration of blood cells was high (five cases). In these cases, a stronger UV absorption was observed in the same region where proteins absorb (below 250 nm), the extra absorption being tentatively assigned to the presence of cells. 10. Sjoholm, I.; Sjodin, T. Biochern. Pharm. 1972,21, 3041-3052. 11. Benet, L. Z.; Sheiner, L. B. In The Pharmacological Basis of Therapeutics; Gilman, A. G.; Goodman, L. S.; Gilman, A,, Eds.; Macmillan Publishing Co.: New York, 1980; pp 1675-1737. 12. Regamey, C.; Humair, L. Postgrad. Med. J . 1971, (Suppl., February), 69-78.
Acknowledgments Support of this work by the Direccion General de Investigacion Cientifica y TBcnica (DGICYT), Ministerio de Education y Ciencia (Spain), through grant PB93-0559 is gratefully acknowledged. We thank Prof. Julio D. Martin (University of La Laguna) for his valuable support and interest in this work. P. G. thanks the Excmo. Cabildo Insular de Tenerife (Spain) for a fellowship.
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Journal of Pharmaceutical Sciences / 1321 Vol. 84, No. 11, November 1995