HPLC Method For The Determination Of Diazepam In Dog Plasma And CSF Of Nasal And Intravenous Administration

Background and purpose: Epilepsy is common disease of CNS (central nervous system) and difficult for emergence treatment because there is no prediction. Especially for the status epilepticus, with high rate of mutilation and death, it is the most important to terminate seizure as soon as possible. IV (intravenous injection) of benzodiazepines is the choice drug and administration route for status epilepticus, but it was limited without staff and easy to cause the accumulation in vivo repeated. So it become a study point to looking for a convenient, quick and high-performance route targeting CNS. For the past few years, people pay attention to nasal cavity because of the specific relationship between it and brain in anatomic structure. This experiment investigate the feasibility of IN (nasal injection) of DZ (diazepam) and provide experimental evidence for it become a substitution or choice administration of IN for IV by analyzing and comparing the pharmacokinetics parameter of DZ in blood and CSF (cerebrospinal fluid) between the IN and IV of propylene glycol solution of DZ.Materials and Methods: 4 adult healthy dogs were divided into A, B two groups randomly: The dogs of A group were administered in nasal at first and in vein after 15 days of metabolism time and the dogs of B group were administered in vein at first and in nasal after 15 days. Every dog was collected blood and CSF simultaneously at 2, 5, 8, 11, 15, 30, 45, 60, 90minutes after administration. All samples were detected by HPLC (high performance liquid chromatography) and each AUC (area under the drug concentration-time curve) was calculated by trapezoidal rule. Intra-groupand inter-group were statistical analyzed with delineation and variance analysis (ANOVA) respectively.Results:1. HPLC was achieved at ambient temperature on a Nova-Pak C18(3.9mm×150mm, 4μm)analytical column. The mobile phase consisted of methanol-deionized water in a ratio of 70:30(v/v) for the plasma and CSF samples analysis. The flow rate is 1ml·min-1. UV detection is set at 254nm. Draw the standard curve of DZ in plasma sample and get the regression equation: Y=6.05e+001X-9.96e+002, r=0.9989(n=5), the linear range of DZ is 50~2000ng·ml-1 and the lowest detectable limit is 15μg·L-1 (S/N≥3) for plasma sample, method recovery is 88.55~99.97%, inter- and intra-day variations are less than 5% for plasma sample. Draw the standard curve of DZ in CSF sample and get the regression equation: Y=3.58e+001X-7.06e+002, r=0.9999(n=5), the linear range of DZ is 10~200ng·ml-1 and the lowest detectable limit is 2μg·L-1(S/N≥3) for CSF sample, method recovery is 98.71~101.20%, inter- and intra-day variations are less than 5% for CSF sample.2. The group of intravenous administration: In plasma concentration of DZ achieve peak value instantly after administration and the peak concentration is 1742±110.18ng·ml-1(n=4), the AUCIV-P is 21795±1402.83min·ng·ml-1(n=4); In CSF the peak time of DZ is 5min and the peak concentration is 84.06±14.08ng·ml-1(n=4), the AUCIV-CSF is 3020.38±220.97min·ng·ml-1 (n=4).3. The group of nasal administration: In plasma concentration of DZ achieve peak value instantly after administration and the peak concentration is 1053.75±118.02 ng·ml-1(n=4), the AUCIN-P is 14450.25±560.11min·ng·ml-1(n=4); In CSF the concentration of DZ has double peaks, the first peak time of DZ is 8min and the peak concentration is 84.06±14.08ng·ml-1(n=4), the second is 60min and the peak concentration is 29.30±1.32ng·ml-1(n=4), the AUCIV-CSF is 2395.79±162.43min·ng·ml-1(n=4).4. The absolute bioavailability (FIN= AUCIN-P/AUCIV-P) of DZ nasal administration is 66.56±5.87%(n=4), and the AUCIN-P is lower than the AUCIV-P (P<0.01). The AUCIN-CSF is 79.72±0.09% (n=4) of the AUCIV-CSF and without significant difference between them (P>0.05).5. The CSF permeability index of IV (FCSF-IV=CIV-CSF/AUCIV-P) is 13.88±1.03%(n=4), the highest is 14.83%; The CSF permeability index of IN (FCSF-IN=AUCIN-CSF/AUCIN-P) is 16.58±0.98%(n=4), the highest is17.65%; The CSF permeability index of IN is higher than the IV (P<0.01).Conclusions: 1. HPLC method is ripe, maneuverable, reproducible and sensitive and can be used for detecting varied biological specimen of diazepam; 2. Compared with the IV, IN of DZ have high absolute bioavailability, the peak time of DZ is identical in both plasma. In CSF the peak time of DZ of IN is later than IV, but it can achieve peak value in few minutes and can achieve the similar concentration with IV; 3. DZ is absorbed completely and rapidly and have high permeability of CSF with IN, it can satisfy the requirement of emergency treatment for status epilepticus out hospital.