Address correspondence to Dr. Henry. .... EM, Henry EL, Murayama M. Sickle cell disease: Two new ... Gobert JG, Bakes EL Availability and plasma clearance of.
CLIN.CHEM.
29/4, 664-666
(1983)
Liquid-Chromatographic Quantification of Piracetam Robert N. Nalbandlan,1.2Marc F. Kublcek,3William J. O’Brien,3Bruce Nichols,1Raymond L Henry,1 George A. Williams,3Arnold I. Goldman,3 Denis Adams,1 and C. M. Teng1 Piracetam,an analog of gamma-aminobutyncacid, absorbs maximally at 197 nm. Itsmolarabsorptivityat 208 nm and pH 4.5 is 3576 (SD 251) L moI
cm1, approximately 45% of
its absorptivity at 197 nm. Direct quantificationof piracetam at 197 nm in biologicalextractsis complicatedbythe factthat many other compoundsabsorb between 190 and 220 nm due to carbon-nitrogen bonding.Chromatographyof methanol extracts of serum and aqueous humor on a reversedphaseC-18 columndevelopedisocraticallywithKH2PO4(0.1 mol/L, pH 4.8) allows detection and quantification of 0.2 mmol of piracetam per liter. Under these conditions the
retention time of piracetam is about 5 mm. The detector response islinearforquantities between 5 and 15 nmol.The method is rapid, inexpensive,and convenientfor the clinical laboratory. AddItIonal Keyphrases: drug
assay
reversed-phase
liquid
chromatography Piracetam, 2-oxo-1-pyrrolidine acetamide, a cyclic derivative of gamma-aminobutyric acid, has been used worldwide for more than 15 years for treatment of disorders of the central nervous system (1-4). Recently, several beneficial effects of piracetam on the formed elements of the blood have been reported (5-B), increasing the need for a rapid, convenient,
and inexpensive
quantitative
method
appropri-
ate for use in the hospital clinical laboratory. Although Gobert and Baltes (9) published a gas-chromatographic method intended for research applications, their method is too time-consuming for clinical use. A rapid, sensitive assay is needed to monitor serum and tissue concentrations of piracetani during therapy. Our purpose here was to develop such an assay for piracetam, not only in serum but (because of some special interests) in aqueous humor as well.
Materials and Methods Sample preparation: Working solutions of piracetam were prepared freshly each day by dissolving weighed amounts of
drug in doubly-distilled de-ionized water. The final concentration was calculated from the absorption at 208 nm according to Beer’s law. Routine ultraviolet absorbance profiles of piracetam were recorded with a Model 25 Kinetice System with automatic wavelength scanner (Beckman Instruments, Fullerton, CA 92634).
1 Department of Physiology, School of Medicine, Wayne State University, 540 E. Canfleld, Detroit, MI 48201. 2Pnt address: Chief Department of Laboratory Medicine, Al Baha King Fahed Hospital, Al Baha, Saudi Arabia. 3Department of Ophthalmology, Eye Institute, The Medical College of Wisconsin, Milwaukee, WI 53226. Address correspondence to Dr. Henry. Received Aug. 23, 1982; accepted Jan. 13, 1983.
664 CLINICALCHEMISTRY, Vol.
29, No.
4, 1983
Whole blood from normal human volunteers or anesthetized (ketamine hydrochloride, 100 mg/kg body weight) New Zealand White rabbits was collected and the serum was isolated. Primary aqueous humor of rabbits was obtained by paracentesis with a 26-gauge needle and 1-niL syringe.
Samples
containing piracetam were combined with two of methanol, vortex-mixed, and let stand at room temperature for 15 miii. The samples were then vortexmixed again and centrifuged (10 000 x g, 10 mm) at room temperature. The supernate was decanted and filtered by centrifugation through a Centriflo CF 25 membrane ifiter (Amicon Corp, Lexington, MA 02173). Aliquots of the methanol-water extract were then directly injected into the chromatograph. Piracetam concentrations were calculated volumes
by peak-height analysis. Chromatography: Although the absorption piracetam is at 197 nm (molar absorptivity
maximum of at 197 am = 7950 L mo11 cm), the optics of most spectrophotometers are reliable only in the range of 200-700 nm. Accordingly, we selected 208 nm as the absorbance wavelength for routine detection of piracetam (10). At this wavelength the molar absorptivity is 45% of that at 197 am (a = 3576 ± 251 L mo11 cm’, n = 10, pH 4.0-5.0). The “high-performance” liquid-chromatographic (HPLC) system we used was a Beckman Model 344 liquid chromatograph, including Model 112 pumps and a Model 165 detector with a Beckman analytical flow cell (1-cm pathlength), operated at room temperature (20 “C). Samples were injected onto the column with a Beckman Model 210 loop injector. Chromatograms were recorded with a Model BD4O recorder (Kipp and Zonen, Deift, The Netherlands). In all cases, a 3.5 x 3.9 mm guard column packed with Bondapak CiWCorasil (Waters Associates, Milford MA 01757) was used. The analytical column was a 30 cm x 4.0 mm (i.d.) stainlesssteel column prepacked with Bondapak C15 from Waters Associates. Samples were eluted isocratically with 0.1 mol/L KH2PO4, adjusted to pH 4.8 with 0.1 molIL HC1, filtered through a 0.2-Lrn (av. pore size) filter, and degassed under reduced pressure for 15 mm before use. The flow rate was 1.5 mL/min and the detector was set to measure absorbance at 208 am. All chemicals and solvents, including water, were HPLC-grade OmniSolv MCB reagents (Curtin Matheson Scientific, Houston, TX 77001). .
-
Results HPLC analysis of piracetam solutions from three different lots produced a single major absorbance peak with a retention time of 5.04 ± 0.01 miii. The molar response was linearly related to amount of sample between 5 and 15 nmol (Figure 1, A and B). Two minor contaminants observed in lot 3010 represent
