Simultaneous Determination of Chlordiazepoxide and Its N-Demethyl Metabolite in 50-pL Blood Samples by High Pressure Liquid Chromatography Hebe B. Greizerstein" and Caryn Wojtowicz Research Institute on Alcoholism, New York State Depa~mentof Mental Hygiene, 1021 Main Street, Buffalo, New York 14203
A method for the determination of chlordiazepoxide and Its Ndemethyl metabolite in 50-yL samples of mice blood by high pressure liquid chromatography has been developed. The drugs together wlth an Internal standard (chlorpromazine) were extracted from alkalinized whole blood with heptane containing 1.5% isoamyl alcohol. The separation was achieved by the use of a solvent gradient program where the eluant was a mixture of 1 mM KH2P04(pH 8.0) and methanol. The method was reproduclble at levels ranging from 0.1 to 10 pg/mL and it is routinely possible to detect as little as 0.1 pg/mL of the compounds in a 50-yL sample. The use of a small volume of sample allowed for the serial sampling of blood from the same animal. The method was tested to determine the concentration of these substances in blood of mice inJected ip with a 30 mg/kg dose of chlordiazepoxide.
There is a n increasing need for sensitive and precise methods for measuring the concentration of chlordiazepoxide (7-chloro-2-methylamino-5-phenyl-3H1,4-benzodiazepine4-oxide) and its N-demethyl metabolite (7-chloro-2-amino5-phenyl-3H-1,4-benzodiazepine-4-oxide) in blood and biological fluids both for research purposes and for monitoring therapy. Several methods using colorimetry ( I , 2 ) , fluorometry ( 3 ) , gas chromatography (4-8) and differential pulse polarography (9) have been reported. These methods are directed specifically toward the determination of chlordiazepoxide and are not suited for small volume samples. Work in our laboratory required a microscale method for accurate measurement of chlordiazepoxide and its N-demethyl metabolite levels in serial small samples of blood from a single mouse. This report presents a simple and accurate method of quantification using reverse phase high pressure liquid chromatography and t h e addition of a n internal standard. EXPERIMENTAL Apparatus. The analyses were carried out in a Waters Associates Liquid Chromatograph equipped with a Model 440 Absorbance Detector (254 nm fixed wavelength), a dual pumping and solvent programmer system, a U6K Universal injector, and a 10-mV recorder. The separation of the drug and its metabolite was achieved using a 4 mm i.d. X 30 cm p Bondapak CIScolumn (Waters Associates) and the programmed elution of solvent (curve 2) a t a flow rate of 2 mL/min. The eluant used was a solution consisting of 1mM KH2P04buffer (pH 8.0) and methanol a t an initial composition of 60:40 v/v that changed to 10:90 v/v final solution in a 3-min period. After the completion of a run, the program was reversed and an additional 2-min period was required for the column to stabilize before the next injection was made. Reagents. Methyl alcohol was acquired from Burdick & Jackson; all the other chemicals used were reagent grade. The drug standards were obtained from Applied Science Laboratories (chlorpromazine, chlordiazepoxide) and Hoffmann-LaRoche (N-demethyl chlordiazepoxide). Sensitivity a n d Linearity Response of t h e Detector. Standard solutions were prepared by volumetric dilution in methanol of stock solutions of the drugs to produce concentrations of chlordiazepoxide and N-demethyl chlordiazepoxide in the range
of 0 to 5 pg/mL. The concentration of chlorpromazine (internal standard) was kept constant a t a 1 pg/mL level. Aliquots of the samples (20 pL) were injected in duplicate into the chromatograph. Recovery. Whole blood standards were prepared by adding to a series of 50-pL drug-free blood samples, 10 pL of methanol solutions containing different amounts of the compounds of interest. The final concentrations achieved were in the 0.1 to 10 pg/mL range. These standards were extracted as in the procedure for blood samples. Extraction Procedure. To 50 pL of blood in a Teflon lined, screw capped test tube, 10 WLof a 50 pg/mL chlorpromazine HCl methanol solution (internal standard) was added. The pH of the mixture was adjusted to 9.0 with 125 pL of 0.1 N NaOH. The extraction was carried out by the addition of 3 mL of heptane containing 1.5% isoamyl alcohol. The mixture was vortexed for 2 min and centrifuged on a clinical bench centrifuge for 15 min. The organic layer was removed to a second test tube and evaporated to dryness under a gentle stream of nitrogen. The dried extract was reconstituted with 100 pL of methyl alcohol, vortexed, and 20-pL aliquots were injected twice into the liquid chromatograph. At the beginning of our studies, we used phosphate buffer (pH 9.0) instead of NaOH to achieve the right pH in the aqueous solution previous to its extraction with the organic solvent. The chromatograms of those extracts showed interfering peaks that made difficult the identification and quantitation of the compounds of interest. Presently, we do not have an explanation on the nature of those interferences. D r u g Blood Levels in Mice. A group of 15 male CF-1 mice 25-34 g in weight, were injected ip with 30 mg/kg chlordiazepoxide HC1 in isotonic saline solution. Tail vein samples, each 50 pL in volume, were obtained only twice from each mouse allowing an interval of 2 h between samples. The time periods studied were 10, 30, 60, 120, and 180 min and 5 blood samples were collected for each point. A parallel set of whole blood standards was prepared by adding to drug-free blood known amounts of chlordiazepoxide and N-demethyl metabolite in methanol to produce concentrations in the range of 0 t o 10 pg/mL. The blood standards and samples were extracted simultaneously. The concentrations of the compounds in the blood samples were determined using the blood standards as the calibrating solutions. This procedure minimizes the differences in recovery rates. Calculations. Peaks on the chromatogram were identified by their retention times relative to the internal standard. The concentration of the drugs was calculated on the basis of the ratio of their peak heights to that of the chlorprcimazine internal standard. RESULTS AND DISCUSSION S e n s i t i v i t y and L i n e a r i t y . T h e detection limit for chlordiazepoxide and its N-demethyl metabolite is 2 ng of compound injected in the chromatograph. T h e response curves for chlordiazepoxide and N-demethyl chlordiazepoxide were found t o be linear (y = -0.024 + 0.596 x and y = -0.171 + 0.935 x , respectively) from the limit of detection near 2 ng to the higher sample size studied. T h e correlation coefficients calculated for the regression lines were 0.99 and 0.97, respectively. Using a sample size of 50 pL of blood, the drug and its metabolite were readily quantitated t o 0.1 pg/mL. Recovery. These data were calculated by the amount of drug detected after extraction of blood samples t o which t h e pure compounds were added in relation to the concentration ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
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Time (hours) Figure 2. Drug concentration curves in blood of mice after administration ip of 30 mg/kg chlordiazepoxide. Each point represents the mean of 5 samples f S.E.M.
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Time (Minutes) Figure 1. Chromatogram of the extract of a blood sample taken from a mouse injected with chlordiazepoxide of these pure compounds in methanol solutions. Recovery was found to be 89 f 3, 68 f 2 , and 94 f 4 % for chlordiazepoxide, N-demethyl chlordiazepoxide, and chlorpromazine, respectively. Drug-Blood Levels in Mice. A characteristic chromatogram of a blood sample taken 10 min after the administration of chlordiazepoxide is shown in Figure 1. The peak appearing between the chlordiazepoxide and chlorpromazine peaks was due to the solvent program used for the separation. The run was completed in 20 min and interfering peaks were not observed in the series of blood samples. Calibration curves were calculated from the set of blood standards, 0.014 + 1.27 x , and -0.023 + 119 x for the drug and the N-demethyl metabolite. Although chlorpromazine eluted after the large peak because of some impurity, the reproducibility of the results provides enough justification for its use as the internal standard. T h e concentrations of drug in blood of mice injected with chlordiazepoxide solutions are represented in Figure 2. Chlordiazepoxide was not detected in the samples taken 2 and 2236
ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
3 h after administration of the drug, while the N-demethyl metabolite had reached a plateau level after 30 min. We have attempted the identification of demoxepam, another of the metabolites of chlordiazepoxide. Under the described chromatographic conditions, this substance has a shorter retention time and similar limit of detection as Ndemethyl chlordiazepoxide. However, its recovery from blood by the extraction procedure here presented is very low (
