Methods For Determination Of Morphine Using High-Performance Liquid Chromatography And Studies On Carrier Erythrocyte Encapsulated Morphine

In order to explore possibilities of carriers erythrocyte encapsulated morphine(M-RBC) by a hyperosmotic resealing method in clinical analgesic therapy. more basic and clinical studies are essential.1. Development and evaluation of methods for determination of morphine (M) in plasma and free morphine in vitro by high-performance liquid chromatography (HPLC)A highly sensitive liquid chromatography method for determination of morphine in plasma was described. This method was based on the selective reaction of mophine with benzylamine in the presence of potassium hexacyanoferrate(Ⅲ) to give a highly fluorescent derivative. The derivative was determined by isocratic elution using 50mM ammonium perchlorate in water-methanol (35∶65,pH3.75) on a C18 reversed-phase column. Dipyridamole was used as internal standard. The detection limit for M was 1ng·mL-1 in plasma . The calibration graph was linear from 1 ng·mL-1 to 1000 ng·mL-1 in plasma. The average recovery was 99.6% and RSD was 3.9%. HPLC with ultraviolet detection (HPLC-UV) was developed for the determination of free morphine in vitro. Morphine is separated on an C18 analytical column. 0.1% tyiethylamine in water-methanol (75∶25,pH7.0) was used as mobile phase. The wavelength of detection was set at 283nm, Linearity was verified from 1 to 50μg·mL-1. The average recovery of morphine 100.8% and RSD was 3.1%. The detection limit for M was 20 ng·mL-1. This method is simple, rapid and accurate for quantitative analysis of free M in vitro.2. In vitro studies on M-RBC by a hyperosmotic resealing method.Mean cell volume (MCV) of dehydrated-RBC was less than native-RBC, while MCV of M-RBC was bigger than native-RBC. M-RBC exhibited a tendency toward spherical shape and dehydrated-RBC exhibited a tendency toward flat shape. Vacuolization and Shrinkage were found in partial dehydrated-RBC and disappeared when M-RBC were achived. About 1/3 Hemoglobin overflowed from RBC. The osmotic fragility cures (OFC) of native-RBC, dehydrated-RBC and M-RBC all displayed S Shape and indicated they had similar resistance to hypotonic heamolysis. Encapsulation yield of M-RBCs achieved by fresh banked blood, human blood and rabbit blood were 37.1%±11.3%,31.4%±11.9% and 20.5%±8.3%,respectively.3. Pharmacokinetics of morphine loaded into erythrocyte in rabbit The cross-over design was used to study morphine pharmacokinetics in 6 rabbits after a single iv dose. Treat group were treated by M-RBC. Controls were treated by free M. Blood samples were collectted at different time after administration and determined drug concentration in plasma. Parametes were as following: AUC and T1/2 of the treat were greater than that of the control but clearance of the treat was lower than that of the control(P<0.05). The result suggested that M-RBC may be an acceptable controlled-release system.4. Preliminary clinical trials on M-RBC Analgesic efficacy were investigated in a clinical study of 40 operation patients after a single iv dose with either M-RBC (treat group) or free M (control group). Compared with the control, there was a significant reduction in pain intensity in the treat in 24h after operation. 90% of the control suffered severe pain and must be treated by synlandine, which was higher than that of treat group. The time from administration M or M-RBC to the first treatment by synlandine was statistically significant (P<0.05). The plasma samples were collected at different time of 10 patients in treat and determined M concentration in plasma.T1/2, 6.48h±1.56h, was greater than that of the reported. These results suggested that M-RBC may as an substitute to the administration of free M in the clinical field.