A sensitive GLC-ECD method for determination of metoclopramide in biological fluids
Creators: name: Tam, Yun Kau
Publisher: name: University of British Columbia
Year: 2010

description: Metoclopramide, 4-amino-5-chloro-2-methoxy-N-(2-diethyl-amino-ethyl) benzamide, a procaine derivative with antiemetic activity, is currently-used in gastro-intestinal diagnostics and in the treatment of various types of gastro-intestinal disorders. Metoclopramide increases the tone and peristalsis of the stomach and duodenum, distends the duodenal bulb and improves the pyloric activity, thus promoting gastric motility and reducing gastric emptying time. This effect of metoclopramide has led to the modification of the absorption of other drugs when administered concomitantly (e.g. digoxin, griseofulvin, the salicylates, and levodopa.) Despite the pharmacological and therapeutic knowledge accumulated in the literature, little pharmacokinetic information pertaining to this drug is available. This is due, in part, to the lack of a highly sensitive assay to quantitate trace amounts of the drug in small volume biological samples. This thesis reports a highly sensitive GLC-ECD assay which is capable of detecting picogram amounts of metoclopramide in the biological fluids in rats. This method involves extraction, derivatization, removal of excess derivatizing agent and quantitation by injecting the derivative solution into a Reporting GLC-ECD. The structure of the derivative was confirmed by using GC/MS electron impact and chemical ionization methods. The optimized conditions are described as follows: Purification - After extraction and drying, the free base was recrystallized three times with, benzene. The crystals were dried at 125°C under vacuum for 2 hrs. This process removed any volatile substances such as benzene. The purity of the free base was confirmed by differential scanning calorimetry. Extraction - Pesticide grade benzene was found to be the best solvent for extraction due to its high purity and satisfactory extraction efficiency for the drug after alkalinization of the aqueous layer with 1 ml of 1 N NaOH. The optimum aqueous and organic ratio for extraction was 1 to 3, viz., 2 ml of the aqueous layer was extracted with 6 ml of benzene. Extraction was accomplished by shaking the aqueous and the organic mixture on a wrist-action shaker for 20 minutes. After centrifugation, 5 ml of the organic phase from a sample was pipetted into a separate 15 ml centrifuge tube. One ml of diazepam solution (750 mcg/ml in benzene) was added to each tube. The content was dried under a gentle stream of nitrogen. The residue was reconstituted with 1 ml of benzene. Derivatization - Twenty ul of heptafluorobutyric anhydride was added to the reconstituted sample. The reaction mixture was incubated at 55°C for 20 minutes. Removal of excess derivatizing agent - The removal of the excess derivatizing agent was achieved by hydrolysis with 0.5 ml of water and neutralization with 0.5 ml of 4% ammonium hydroxide solution. GLC - One ul of the derivative solution was injected into a GLC-ECD with the following conditions: injection temperature: 250°C oven temperature: 250°C carrier gas(95%/5% argon/methane) flow rate: 40 ml/ min. A 1.8 m, 2 mm i . d . glass column packed with 3% OV-17 coated on Chromosorb W (80-100 mesh) was used. GLC/MS - The structure of the HFB derivative was elucidated by GLC-electron impact mass spectrometry. The following conditions were used: injection temperature: 250°C column temperature: 250°C carrier gas (helium) flow rate: 40 ml/min A 1.8 m, 2 mm i.d. glass column packed with 3% 0V-17 coated on Chromosorb W (80-100 mesh) was used. ionization beam energy: 70 eV electron multiplier voltage: 2 Kv analyser temperature: 50°C separator oven temperature: 200°C Since the molecular ion was not readily discernable from the electron impact mass spectrum, chemical ionization mass spectrometry was used to identify the molecular ion. The following conditions were used: probe temperature: 200°C source temperature: 150 C ionization voltage: 70 V ionizing gas: isobutane samples were introduced by direct probe method Quantitative studies - A calibration curve was prepared for the plasma extracts. Linearity was observed in the range studied (91-750 ng/ml). The recovery of drug spiked separately into plasma, whole blood and urine was 85%. This assay is extremely sensitive and specific. The lowest detectable amount of metoclopramide i s 1 pcg. Animal studies - The applicability of this assay technique was shown by studying the elimination kinetics of the drug in the rat. The time course of the drug elimination after an i.v. dose can be described by a biexponential equation. The half-life calculated was 49.75 - 8 minutes.